Ligand-biased signalling could have a significant impact on drug discovery programs in the pharmaceutical industry. As such, many investigators and screening campaigns are now being directed at a larger section of the signalling responses downstream of an individual G protein-coupled receptor. Biosensor-based platforms have been developed to capture signalling signatures. Despite the ability to use such signalling signatures, they may still be particular to an individual cell type and thus such platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. We have developed a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin arsenical dyes engineered into different positions in the AT1 angiotensin II receptor. We discuss the design and optimization of such sensors to capture ligand bias and cell context-specific effects on receptor conformation.
Retinitis pigmentosa (RP) is a degenerative, inherited eye disease that leads to severe vision loss and blindness at an advanced age. RP is associated with point mutations in rhodopsin, most of which affect the ability of the protein to (i) bind retinal, or (ii) fold and traffic properly or (iii) activate transducin. However, there are certain unclassified mutations of rhodopsin that cause no obvious cellular defects, yet result in RP. Especially interesting for us became the lipid facing mutations F45L, V209M and F220C since Rakoczy, et al. (J Mol Biol 2011) suggested via in in-silico analyses that these mutations would have no obvious problems.
Since rhodopsin was recently identified as a phospholipid scramblase (Menon, Curr Biol. 2011; Goren, Nature Commun. 2014) and given the fact that retinopathies are often linked to lipid trafficking, we considered that some of these unexplained mutations might affect rhodopsin’s scramblase activity. We expressed F45L, V209M and F220C in HEK293S GnTI- cells and assayed the purified proteins for their scramblase activity after reconstitution into large unilamellar vesicles. While all three mutant proteins showed wild type-like scramblase activity, our analyses revealed that they functionally reconstituted into vesicles as monomers unlike the wild-type protein which reconstituted as a dimer. We further characterized those mutants by taking retinal spectra, G protein activation data, retinal entry and exit measurements and fluorescence micrographs.
Ion channels play a vital role in numerous physiological functions and drugs that target them are actively pursued for development of novel therapeutic agents. Here we report a means for monitoring in real time the conformational changes undergone by channel proteins upon exposure to pharmacological stimuli. The approach relies on tracking ligand induced structural rearrangements by monitoring changes in bioluminescence energy transfer (BRET). To provide proof of principle for this approach, we worked with Kir3 neuronal channels producing a raft of 20 different BRET biosensor options. Among these combinations, pairs bearing the donor Nano-Luc (NLuc) at the C-terminal end of Kir3.2 subunits and the FlAsH acceptor at the N-terminal end (NT) or the C terminal domain (CTD-BDBE) of Kir3.1 subunits were identified as potential tools. These pairs displayed significant changes in energy transfer upon activation. Conformational changes associated with channel activation followed similar kinetics as channel currents. Dose response curves generated by different agonists in FlAsH–BRET assays displayed similar rank order of potency as those obtained with conventional BRET readouts of G protein activation and ion flux assays. Furthermore, these biosensors allowed for us to measure interactions not only with channel activators (directly, or via the d-opioid receptor (dOR)) but also with otherwise silent ligands of the Kir3 channel, such as the channel blockers BaCl2, Ethosuximide and Tertiapin Q that interact directly with the Kir3 channels, but do not cause activation, and cannot be discerned by conventional screening unless agonists are present. Conformational biosensors as the ones reported here should prove a valuable complement to other methodologies currently used in channel drug discovery.
Recent advances in the structural biology of G protein-coupled receptors (GPCRs) have unraveled many intricacies underlying hormone and ligand binding. Delineation of the structure of the β2-adrenergic receptor (β2AR) in a complex with the stimulatory G protein Gs has provided a snapshot into the principle step that precedes GTP binding and G protein activation, GDP release. These data and more recent structural analyses of the muscarinic M2 and mu-opioid receptors in their activated state also reveal a possible mechanism through which G proteins in turn allosterically modulate hormone binding. We have utilized camelid antibodies (nanobodies) generated against various agonist-bound GPCRs as well as antibodies generated against the β2AR•Gs complex to assess the underlying mechanism of G protein activation as well as the modulatory role that G proteins play on agonist binding. Here we also take advantage of G protein nanobodies to study the G protein side of the β2AR•Gs complex. One of the more surprising aspects of the β2AR•Gs complex revealed by both x-ray crystallography and electron microscopy is the major conformational change in Gsα. The structure reveals a dramatic rigid-body translation and rotation of the helical domain away from the nucleotide-binding ras domain of Gsα. To investigate the relationship between domain opening and nucleotide exchange we apply a series of computational and biophysical methods as well as utilize G protein nanobodies to stabilize the open conformation. Data obtained from these studies suggest that domain opening occurs spontaneously and is a critical requirement for GDP release.
The family of adhesion G protein-coupled receptors (aGPCRs) facilitates cell-cell and cell-matrix interaction. GPR56 is a member of the aGPCR family and its mutations are responsible for a human brain malformation known as bilateral frontoparietal polymicrogyria (BFPP). Individuals with BFPP present with defects in central nervous system (CNS) myelination, in addition to cortical malformation. However, the cellular role of GPR56 in oligodendrocyte development remains unknown. Using mouse and zebrafish as model organisms, we demonstrated how GPR56 and its ligand regulate oligodendrocyte development and CNS myelination.
In the last decade, crystallisation of membrane proteins in lipidic cubic phases (LCP; in meso) boosted the numbers and resolution of membrane-protein structures. However, membrane-protein crystals are usually small and notoriously difficult to harvest from the highly viscous LCP. Recently, an in situ in meso approach has been introduced, in which crystals are not harvested and flash-frozen but placed in the X-ray beam within the mesophase at room temperature. In this study, we introduce novel in situ in meso plates that show significantly less background scattering, are easier to handle, and are variable with respect to the crystallisation volume. Moreover, we developed holders for either individual or up to four in situ wells at a time, which are easy and cheap to produce, easy to handle, reusable, and compatible with measurements at room temperature and under cryogenic conditions. Because the holders are attached to standard ALS-type goniometer bases, they allow for storage and shipping of entire wells (with typically several dozens of crystals) in Universal Pucks under liquid nitrogen and for auto-mounting at synchrotrons. We validated the new setups using water-soluble hen egg lysozyme and the membrane protein bacteriorhodopsin. In conclusion, this study demonstrates the potential of combining in meso crystallisation with in situ diffraction and the possibility to store, ship, and measure crystals under cryogenic conditions for obtaining structural information on membrane proteins. In conjunction with the current developments at synchrotrons like smaller beams, faster detectors, and software for multi-crystal strategies, this approach promises high-resolution structural studies of membrane proteins to become faster and more routine.
While preterm labor remains the main cause of infant mortality in industrial world, the need to develop better tocolytics with less side-effects remains. Moreover, a clearer understanding of the prostaglandin F2α (FP) receptor mechanism of action, which is an important mediator of parturition and uterine cell signals leading to contraction, is needed. We previously demonstrated that myometrial cell contractions could be stopped in vitro and in vivo using a peptidomimetic compound, PDC113.824 (PDC), whose structure was originally derived from the second extracellular loop (ECL2) of the FP receptor. This compound selectivity inhibited PGF2α-mediated Rho/Rock signaling cascade via Gα12, while enhancing ERK1/2 and PKC signaling via Gαq, consistent with an allosteric, biased mode of action on FP receptor. This research ought to characterize the mechanism(s) of action by which PDC modulates FP receptor signaling, as well as to define its binding site(s). New molecules, where the indolizidine group of PDC was replaced by various pharmacophores were derived, hence allowing structure-function study. Their effects were tested in myometrial cells that express endogenously FP. When compared to PDC, the derivatives showed differential behaviors on PGF2α-mediated calcium mobilization, MAPK signaling and myometrial cells proliferation, where some potentiated, while other inhibited the effects of the agonist. To better understand how these allosteric ligands bind to FP receptor, we introduced photoactivable moieties in their structures with different chemical constraints. We first assessed their activities on PGF2α-mediated MAPK. Altogether, the use of these modulators should allow better understanding of how FP allosteric modulators function and help developing more efficient tocolytics.
This work is funded by CIHR and March of Dimes (SAL), and a Pharmacology and Therapeutics studentship award (LN).
Regulator of G-protein signaling 2 (RGS2) is known to inhibit Gq- and Gs-mediated GPCR signalling. We have previously reported that RGS2 knock out mice (rgs2-/-) exhibit a lean phenotype compared to their wildtype (WT) counterparts, with significantly reduced fat deposits, leptin levels, and serum lipids. The objective of this study is to further elucidate the role of RGS2 in metabolism. CLAMS metabolic cages were used to measure various indices in 1 month-, 3 month-, and 12 month-old rgs2-/-and WT mice. Animals were acclimatised in metabolic cages for 24 hours (12-hour light and dark cycles), followed by another 24 hours of metabolic recordings. At both 3 months and 12 months, rgs2-/- mice displayed increased rates of respiratory intake (vO2) and expiratory output (vCO2), particularly during the dark cycle, suggesting increased metabolic activity in the knockouts. Furthermore, calculated respiratory exchange ratios (RER) suggest an increase in carbohydrate rather than fat or protein metabolism in rgs2-/-mice of all three age groups compared to WT. In addition, hyperphagia, as well as increased water intake, were also observed in the knockouts at 3 and 12 months. Even with significantly increased food and water intake, 3 and 12 month-old mice maintained lower body weights compared to WT mice, with 3 month-old mice displaying increased ambulatory activity during their dark cycles. In addition, a 30% diet restriction study followed by metabolic cage measurements was performed to determine possible alterations in metabolism following changes in food intake. Compared to our previous ad libitum diet studies, 3 month-old rgs2-/-mice had significantly higher vO2 and vCO2 rates, particularly during the light cycle. Furthermore, the RERs of rgs2-/-mice were significantly higher than WT, and seem to confirm elevated carbohydrate metabolism in rgs2-/-mice. Interestingly, diet restriction appears to have exacerbated the hyperphagic phenotype of rgs2-/-mice – although body weights remained stable, rgs2-/-mice consumed 50% of their daily food amounts at a 4 times higher rate than WT mice, and displayed hoarding behaviour. These results suggest that RGS2 may play an important role in the regulation of body metabolism.
Introduction: Stimulation of delta opioid receptor (DOR) by agonists stimulate multiple pathways which lead to analgesia. However chronic administration of opioid agonists leads to tolerance, and reduced therapeutic effect. A correlation has been found in the literature (Pradhan et al. 2009) between drug efficacy to different cellular/molecular response and tolerance for DOR agonists. The goal of our study is to have a better understanding of the phenomenon of tolerance from a pharmacological viewpoint. To this end, we have quantitatively investigated the correlation between analgesic efficacy of different analgesic DOR ligands and the development of chronic tolerance. Methods: Systemic injection of streptozotocine in rats will induce type 1 diabetes. We evaluated analgesic efficacy in this diabetic rat model of neuropathic pain over one week. Our aim was to assess homologous tolerance induced by 4 different drugs (SNC-80, deltorphin II, TIPP and SB 235863) that were administrated by intra-thecal (i.t) injection (ED80) to different groups of animals. Tolerance was evaluated by measuring mechanical allodynia after injection of each drug over days 2-7. Before starting the chronic treatment by 4 different agonists, a challenge dose of deltorphin II (ED50) was administrated on day 1. On the last day (day 8), the challenge dose of deltorphin II was re-injected to assess heterologous tolerance. Results: At the end of the chronic treatment with peptidic agonists (deltorphin II and TIPP), the analgesic effect of the ED50 dose of deltorphin II was not different from that observed on day one (pre-prolonged drug treatment). However, prolonged treatment with non-peptidic agonists (SB and SNC-80) reduced the effect of this second of deltorphin II injection by 60%. Both of these agonists also induced homologous tolerance but SNC-80 (t1/2 ~ 1 day) did so faster than SB235863 (t1/2 ~ 3 days). Homologous tolerance for deltorphin II was not observed and TIPP maintained half of its analgesic effect. Conclusion: The tolerance pattern was not related to analgesic efficiency of the drug, as measured by the operational model nor by internalization profiles of the drugs, which had both been previously proposed as predictor of analgesic tolerance. The only systematic difference observed was by peptidic agonists (reduced or no tolerance) and non-peptidic agonists (produces tolerance) ligands.
Heart failure is a major public health concern. Increased risk of heart failure is significantly associated with cardiac hypertrophy. Cardiac hypertrophy is characterized by the hypertrophic growth of cardiac myocytes, driven in part by increased levels of neurohumoral agonists such as endothelin acting on myocyte G-protein coupled receptors (GPCR). Recently, our laboratory showed that nuclear envelope scaffolded form of phosphoinositide-specific Phospholipase C (PLC), PLCε, responds to Plasma membrane (PM) endothelin 1 receptor stimulus to hydrolyze phosphoinositol 4-phosphate (PI4P) in the perinuclear Golgi apparatus to locally generate diacyglycerol and activate a nuclear pool of PKD in neonatal cardiac ventricular myocytes (NRVM). This signaling process leads to reactivation of the fetal gene program in NRVM (Zhang et al. Cell, 2013). We have also shown that the PM endothelin-1 receptor (ET-1AR) stimulation of Golgi PI4P depletion and nuclear PKD activation is dependent on Gβγ dimer (Malik et al. Mol Biol Cell, 2015). However, how activation of ET-1AR at the PM leads to Gβγ signaling at the Golgi remains an open question. Other groups have shown that the Gβγ dimer translocation to the Golgi on GPCR activation is dependent on the Gγ isoform. The different translocation property of the Gβγ dimers is determined by variations in the basic and hydrophobic residues in the Gγ subunit C–terminal domain. We reexamined Gγ3, Gγ7 and Gγ9 mediated Gβγ dimer translocation on Gq coupled receptor stimulation in Hela cells. We also attempted to abolish Gγ3 translocation completely by creating Gγ3 mutants that have extra hydrophobic residues at the C-terminal. We observed that the Gβγ dimers containing Gγ9 translocated rapidly to the Golgi compared to those with Gγ7 or Gγ3, and Gβγ containing Gγ3 was the slowest. Moreover, we found no difference in the translocation property of the Gγ3 mutants to wild-type Gγ3. However, since we have confirmed that Gβγ3 translocates slowest, we hypothesize that overexpressing Gγ3 in NRVMs will reduce translocation of Gβγ to the Golgi, inhibit PM endothelin receptor stimulated Golgi PI4P hydrolysis and block cardiac hypertrophy. The result above and results from testing the foregoing hypothesis in NRVMs will be presented.
Pre-term birth, defined as birth before 37 weeks of gestational age, has been identified as one of the most important maternal and child health problems worldwide. Pre-term birth rates available from some developed countries show a dramatic rise over the past 20 years, and accounts typically for more than 75% of all neonatal deaths. This high incidence rate contrasts with the fact that there are no clear first-line tocolytic drugs that delay or prevent pre-term labour. Better pharmacological interventions are needed to act at points foregoing the onset of pre-term labour and upon key factors underlying uterine contractions, such as the prostaglandin F2α (PGF2α) and its G protein-coupled receptor (GPCR), the prostaglandin F receptor (FP). The existence of GPCR heterodimers was recognized more than 15 years ago, and such assemblies can be viewed as signalling hubs or scaffolds for specific hardwired signalling complexes. We hypothesized that the heterodimer formed by FP and the angiotensin II (AngII) type 1 receptor (AT1R), a GPCR with critical implications in cardiovascular homeostasis, might be assembled to control signalling outcomes allosterically. We aimed to provide evidence for formation of such a heterodimer and to assess allosteric control of signalling (J Biol Chem. 2015;290(5):3137-48). One interesting feature we observed in the interplay between FP and AT1R resided in both asymmetrical (i.e., only one of the two receptors modulate the properties of the other) or symmetrical (i.e., both receptors modulate each other properties) behaviors, depending on the outcome being measured. For example, asymmetrical effects were observed when ERK1/2 signalling was examined in vascular smooth muscle cells. FP signalling through ERK1/2 was strongly potentiated by L158,809 (AT1R antagonist) whereas AT1R activation of ERK1/2 was unaffected by AS604872 (FP antagonist). Symmetrical effects also were observed in abdominal aortic ring contraction experiments. PGF2α-dependent activation of FP potentiated AngII-induced contraction whereas the AS604872 had the opposite effect. Similarly, PGF2α-mediated vasoconstriction was symmetrically regulated by AngII or L158,809. No such effects were observed for either Gq activation and Ca2+ signalling so far. However, new evidence suggests PGF2α-dependent recruitment of β-arrestin 2 that is specific to the heterodimer, but not to FP alone. Taken together, our results suggest that dimerization and allosteric regulation of FP and AT1R should be taken into consideration as a new therapeutic avenue.
Hypertension is a common health problem and a risk factor for further severe complications. Enhanced vasoconstrictor-mediated G protein coupled receptor (GPCR) signaling via Gαq contributes to the disease. Regulator of G protein Signaling (RGS) proteins modulate GPCR signaling via their GTPase activating activity. Among them, RGS2 suppresses vasoconstrictor-mediated phospholipase C/Ca2+ signaling by selectively inhibiting Gαq. Certain RGS2 mutations, found to associate with hypertension, have reduced expression and/or function. While ~1.6% of individuals from the NHLBI exome sequencing project have missense mutations in RGS2, their functional significance is unknown. Among them, 13 mutations found in more than one person will be investigated. Whether or how these mutations affect RGS2-dependent control of blood pressure homeostasis remains to be determined. As a first step, we sought to compare the protein levels and relative protein stability of these mutants in vitro. We utilized Gateway cloning technology to shuttle DNAs encoding RGS2 from an entry vector to different expression vectors for various research purposes. Similar to the already characterized RGS2 mutant, Q2L, we found that the A4V mutation also results in reduced protein levels while maintaining mRNA level and protein stability comparable to wild-type protein, and S3G with trend to have lower protein level. These mutants are upregulated by MG-132, a proteasome inhibitor, suggesting that, like wild-type RGS2, they are degraded via ubiquitin-proteasomal pathway. Determining the functional consequences of RGS2 mutations is essential. Difference in protein levels may affect the ability of RGS2 to inhibit G protein signaling, specifically Gαq in the cardiovascular system. The Q2L mutation was associated with hypertension in a Japanese population and it is in our interest to see whether other mutations may have similar clinical implications. All together they may help us one step closer to understand how RGS2 functions in the cardiovascular system.
Opioid receptor agonists are effective analgesics. Clinically available opioids have been developed for the MOR which are effective in acute but not chronic pain management. MOR agonists also produce severe side effects during treatment like respiratory depression and severe constipation. DOR agonists have been shown to be effective in chronic pain and have an advantage over MORs in that they produce fewer side effects, however they have tendency to produce tolerance. Interestingly, not all DOR agonists produce the same degree of tolerance. The differences have been attributed to the DOR ligands ability to distinctly engage cellular determinants of tolerance such as receptor desensitization, receptor internalization and adenyl-cyclase super-activation. Here we investigated whether simple molecular interactions among DORs and different signaling partners were distinct for different ligands and whether these simple signals were predictive of more complex cellular responses linked to analgesic tolerance. To assess ligand-induced changes in DOR interaction with signaling partners we used BRET-based biosensors for G protein and downstream effector activation and ligand effects were analyzed with operational model. Our results clearly indicate that quantitative treatment of simple signals can be predictive of ligand potential to induce endocytosis and cyclase superactivation but not desensitization. In particular, the potential for endocytosis was correlated with ligand bias to produce B-arrestin (B-arr) recruitment versus G-protein activation, but not by ligand efficiency to recruit B-arr or activate G-proteins. Moreover, ligand potential to produce cyclase super activation was directly correlated with ability to mobilize Ca+2. This data provides proof of principle that it is possible to extract information from simple signaling results to predict more complex responses associated with in vivo analgesic tolerance. This type of analysis should allow us to better identify promising analgesic drug candidates early in the drug discovery process.
Protein structure-based drug design (SBDD) approaches, that have been highly successful with soluble targets such as kinases and proteases, can now be used to discover better GPCR drug candidates, including for the more “difficult” and previously undruggable GPCRs. The ability to apply SBDD approaches to GPCRs has been driven by improvements in the purification, and crystallisation of receptors which had been hampered by their inherent instability. To solve the issues relating to instability, StaR® proteins have been engineered in such a way that they have significantly improved thermostability and a chosen conformational state, which is achieved through the introduction of a small number of point mutations. The increased thermostabilisation allows for the receptors to be readily removed from their native cell membranes and purified in a detergent solubilized form. StaR proteins can be used for biophysical mapping™, fragment screening, antibody generation and crystallisation to yield X-ray structures with both potent and weak ligands. Examples of clinical candidates with better properties and selectivity achieved using a combination of kinetic, thermodynamics and structural analysis will be presented
G protein-coupled receptors (GPCR) are highly dynamic proteins which transmit information by interacting with different ligands that promote the activation of multiple, yet specific downstream outputs. It is widely accepted that this is achieved through the adoption of distinct conformations in the GPCR whereby individual conformational states preferentially drive a specific receptor function. The available conformational capacity, and therefore possible receptor functions, can be further expanded when considering allosteric interactors. Receptor complex formation, such as oligomerization, may induce novel conformational constraints providing mechanisms for new signalling modalities or protein life-cycle behaviours.
Using GPCR conformation-sensitive biosensors, we investigated allosterically-induced conformational changes in the recently reported F prostanoid (FP)/angiotensin II type 1 receptor (AT1R) heterodimer (1). We noted conformational cross-talk between the partner receptors where Ang II activation of the AT1R induced distinct conformational changes in FP when compared to PGF2. This allosteric communication was mediated through Gαq and involves proximal (phospholipase C) but not distal (protein kinase C) signalling events. We are now investigating whether heteromer disruption or receptor signalling affects receptor/receptor mediated conformational cross-talk.
The identification of allosterically-induced GPCR conformations may explain novel receptor functions in such multi-protein receptor complexes.
1. Goupil, E., et al., Angiotensin II Type I and Prostaglandin F2α Receptors Cooperatively Modulate Signaling in Vascular Smooth Muscle Cells. Journal of Biological Chemistry. 290(5): p. 3137-3148.
Heterotrimeric combinations of Gα, Gβ and Gγ subunits interact with G protein-coupled receptors (GPCRs) as transducers modulating a number of complex signalling networks that ultimately converge on the regulation of gene transcription. Gβγ subunits are involved in an array of distinct signalling processes in various compartments of the cell, and of particular interest to our lab, Gβγ subunits modulate the activities of a variety of proteins in the nucleus. We have previously performed ChIP-on-CHIP experiments that have revealed that Gβγ dimers occupy the promoters of more than 800 genes. Our in silico analyses have shown that neither Gβ and Gγ subunits are able to bind DNA on their own, leading to the hypothesis that Gβγ dimers occupy promoters in conjunction with other proteins – transcription factors or proteins involved in the process of transcription. Since Gβγ dimers occupied so many promoters, we assessed whether Gβγ could interact with RNA polymerase II. Here, we demonstrate that Gβγ dimers do indeed interact with RNA polymerase II in an agonist-induced, GPCR-dependent manner. In particular, we have shown that this interaction is induced both by M3 muscarinic acetylcholine receptors in human embryonic kidney cells and angiotensin II type I receptors in primary rat neonatal cardiac fibroblasts. Subcellular fractionation studies showed that upon GPCR activation, Gβγ subunits translocate to the nucleus and interact with Rpb1, the largest subunit of RNA polymerase II. Various inhibitors and cell lines with CRISPR mediated knockout of proteins potentially downstream of GPCR activation were used to decipher the mechanisms that modulate how Gβγ interacts with Rpb1. We will next try and understand the functional consequences of this interaction using ChIP-seq and fibrosis gene arrays in cardiac fibroblasts. Taken together, our studies reveal a novel interaction between Gβγ subunits and Rpb1, further shedding light on the complex roles they play in GPCR signalling.
The eukaryotic Group II chaperonins are composed of eight different subunits forming a pseudosymmetric ring known as the CCT/TRiC complex. This complex was originally characterized to be involved in the folding of newly synthesized actin and tubulin in an ATP-dependent manner, but the list of substrates keeps growing every year. For example, this complex is now known to be involved in the folding of G protein β subunits, WD-repeats proteins and the von Hippel-Lindau tumor suppressor. Recent studies have shown that CCT/TRiC can interact with membrane proteins, such as LOX-1 receptor, and is implicated in the translocation process of PMP22 in the peroxisome membrane. In previous work, we have identified all the eight subunits of the CCT/TRiC complex in a gel-free proteomics approach designed to find partners of the β2-adrenergic receptor (β2AR). We also recently found the subunit eta (CCT7) to be an interaction partner of the β-isoform of the thromboxane A2 receptor (TPβ) C-tail by yeast two-hybrid screening. Co-immunoprecipitation experiments using HEK293 cells overexpressing β2AR or TPβ suggest that there is an association between CCT7 and these receptors without agonist stimulation. Using confocal microscopy, we confirmed that both receptors co-localize with endogenous CCT7 and its depletion re-localize receptors in aggregate-like structures. We observed a diminution of receptor cell surface expression by ELISA when the cells were treated with DsiRNA against CCT7. We identified the domains of interaction on the receptors using His-Pulldown assay and co-immunoprecipitation with deleted receptor mutants. To our knowledge, this is the first demonstration of a functional interaction between the CCT/TRiC complex and GPCRs.
G-protein coupled receptors (GPCRs) are the largest and most diverse family of cell surface receptors and are involved in many physiological processes; as such they make good therapeutic targets. Upon agonist stimulation, GPCRs can signal via G-protein coupling or via a β-arrestin-dependent mechanism leading to receptor internalization. Mitogen-activated protein kinases (MAPK) have been shown to regulate β-arrestins in intracellular trafficking and GPCR signaling. However, the mechanisms by which receptors get internalized remain to be further explored. Here, we contribute to the characterization of a novel compound, Traf 21, which was selected from a high throughput screening for the modulation of Angiotensin II type I receptor (AT1R) internalization.
Using confocal microscopy and bioluminescence resonance energy transfer (BRET) techniques, we show that Traf 21 prevents AT1R internalization in HEK293 cells. Interestingly, Traf 21 has no effect on β-arrestin recruitment to AT1R, but blocks MAPK activation by AT1R. To further characterize the effects of Traf 21 on AT1R function, a structure-activity relationship study was designed and six analogues of Traf 21 were created. Preliminary studies on Angiotensin II-induced MAPK activation and AT1R internalization in the presence of the other compounds show that Traf 21 remains our lead inhibitory compound. Identifying how Traf 21 pharmacologically modulates responses downstream of GPCRs may lead to a better understanding of how receptor internalization is mediated, and the physiological role of such process.
Congenital stationary Night Blindness (CsNB) and retinitis pigmentosa are eye diseases that result in defective night vision and progressive vision loss, respectively. In this study, we recombinantly expressed three rhodopsin disease mutants (G90V, T94I and A295V) in HEK293S GnTI– cells. We combined site-directed spin labeling with Electron Paramagnetic Resonance spectroscopy to characterize the conformational equilibria of purified rhodopsin mutants in both dark and light-activated states.
Using Double Electron-Electron Resonance (DEER) spectroscopy, we monitored rhodopsin’s helix VI and helix VII movements in disease mutants. To do so, we measured distances between two spin probes located at the cytoplasmic ends of helix II and either helix VI or helix VII. We compared DEER distance measurements performed on wild type and rhodopsin mutants solubilized in both dodecyl maltoside and nanodiscs. The latter allowed us to unmask the effects of disease-causing mutations on the receptor conformational equilibrium.
Our data show that rhodopsin disease mutants and wild-type rhodopsin have similar distance distributions in the dark state. However, after illumination, the two CsNB mutants (T94I and A295V) exhibited a greater shift towards the activated state when compared to the wild type. Retinitis pigmentosa G90V mutant could not be fully photo-activated, as shown by its UV-visible spectrum after illumination. This characteristic was partially reflected in the DEER data we obtained in nanodiscs. Indeed, while helix VII motion was comparable to the wild type, helix VI movement seemed to be impaired.
Studying the conformational equilibria of rhodopsin disease mutants in nanodiscs using DEER spectroscopy could bring new insights into the molecular bases of CsNB and retinitis pigmentosa. Also, this approach could be extended to other diseases linked to GPCR mutants.
The protease-activated receptors (PARs), composed of four members (PAR-1, -2, -3 and -4), represent an atypical subfamily of receptor among G Protein coupled receptor (GPCR) family. Indeed, these receptors are activated by the proteolytic cleavage of their N-terminal region by enzymes, such as thrombin or trypsin. This cleavage exposes a region of the N-terminal extracellular domain (called the “tethered ligand”) which can bind residues contained within the second extracellular loop of the PARs, resulting in the stabilization of an active conformation. Short synthetic peptides mimicking the tethered ligand sequence are also able to activate PAR receptors. This subfamily of GPCR is particularly interesting because they are largely involved in inflammatory responses and may therefore represent a promising therapeutic target for the treatment of immune-mediated inflammatory diseases. In this study, we proposed to establish the exhaustive signalling signature of the human PAR-2 in response to trypsin or the PAR-2-selective synthetic peptide (SLIGKV-NH2) stimulation, using BRET-based biosensors. For this, we have stimulated endogenous and overexpressed PAR-2 in two different cell lines, human embryonic kidney cells (HEK293) and human colon cancer cells (HCT-116). Firstly, we determined the Gα protein subunits that are activated by PAR-2 by overexpressing each Gα subunit with the GFP10-GRK2 and Gγ5-RlucII biosensors. Interestingly, we observed that both trypsin and SLIGKV-NH2 activated most of α subunits, including Gq/11, G12/13 and Gi families but not Gs family. We are currently confirming these results using Gα-RlucII and Gγ-GFP10 biosensors. We have next studied signalling pathways downstream of G protein. Consistent with the activation of Gq/11 protein family, we showed that PAR-2 stimulation induced a rapid increase of intracellular calcium and diacylglycerol in both cell lines, associated with an activation of PKC. To evaluate the signalling pathway downstream of Gi and G12/13 families, we are currently investigating the impact of PAR-2 stimulation on cAMP production and GTPase RhoA activation, respectively. Finally, we have also evaluated the recruitment of β-arrestin 1 and 2. Our results showed that PAR-2 recruits both β-arrestin 1 and 2 in response to trypsin and SLIGKV-NH2, in a comparable manner between HEK293 and HCT-116 cells. Altogether, our results allowed a better understanding of signaling profile of human PAR-2 and could therefore contribute to the development of new drugs targeting PAR-2 for the treatment of immune-mediated inflammatory diseases.
Rhodopsin, which absorbs a photon to initiate visual phototransduction, belongs to the superfamily of G protein (guanine nucleotide-binding protein)-coupled receptors (GPCRs). Photoisomerization of the 11-cis-retinal chromophore of rhodopsin triggers a complex set of molecular events leading to light perception. Retinal photoreceptor cells can respond to light throughout our lives because they continuously regenerate a light-sensitive chromophore and certain essential structures. This series of reactions takes place in photoreceptor and the retinal pigment epithelium (RPE) cells. Defects in many proteins involved in these processes cause photoreceptor degeneration. For example, mutations in the rhodopsin gene may cause human diseases like retinitis pigmentosa (RP) that usually result in late-onset blindness. Our long-term goal is to elucidate the molecular mechanisms of phototransduction and retinal degeneration to discover therapeutics for inherited human blinding diseases caused by mutations in phototransduction genes. This is a necessary prerequisite for developing evidence-based therapeutic approaches for treatment of these pathological conditions. Combining disciplines such as state-of-the art imaging, bioinformatics, genomics, and structural biology with classical histopathological, physiological, and biochemical methods can dramatically increase understanding of causes of inherited human retinopathies.
Photosystem II (PSII) in higher-plant chloroplasts is responsible for at least 50% of the oxygen in the atmosphere. It is composed of a reaction center (RC) surrounded by an array of antenna compounds, of which light-harvesting complex II (LHCII) is the most important. Both these complexes consist of chlorophyll pigments held in a specific pattern by a protein matrix. The LHCII captures sunlight and funnels it quickly to the RC. Once there, charge separation occurs, across a special pair of chlorophylls, and evolves into one of the most highly-oxidizing states in nature, with a redox potential > 1 V . This potential drives the water-splitting reaction, in which molecular oxygen is discarded as a side product .
To reveal dynamical information about energy transport routes amongst specific chromophores by probing a complete chloroplast or PSII complex is problematic. Therefore, we extract LHCII and the RC, from spinach. A strong detergent followed by a combination of salts  is used to precipitate out LHCII from its PSII complex solution. Column chromatography then separates the RC from its closely-associated antenna complexes . Care is needed to ensure that the essential molecular machinery and, consequently, the sample dynamics remain intact. The identity, purity, and functionality of the isolated complexes is confirmed with absorbance, circular dichroism, chlorophyll assay, SDS-PAGE gel electrophoresis, and transient absorption measurements.
A plethora of spectroscopic techniques have been used to investigate the early-time (<1 ns) processes occurring in photosynthetic complexes. Of these, two-dimensional spectroscopy is ideal to investigate electronic coupling and charge-transfer dynamics, as it can differentiate overlapping pigment absorption peaks. It has exposed long-lived oscillations, with picosecond durations, between different pigment populations in LHCII [5,6] and the RC , mostly at low temperatures (77 K). These oscillations have been attributed mainly to excitonic coherences that, assisted by fluctuations in the surrounding environment, optimize the energy-transfer and the charge-separation efficiency of the complexes. Recently our own group, studying LHCII at ambient temperature and with a restricted range of excitation wavelengths around its Qy band, has found that there is electronic dephasing in 60 fs, and any oscillations present are vibrational . By using broadband excitation that extends into the vibrational shoulder of LHCII, we will resolve the contribution of vibrations to these oscillations and their implication upon photosynthesis.
 Barber, James. Q Rev Biophys 36.01 (2003):71
 Barros, Tiago, and Werner Kühlbrandt, BBA-Bioenergetics 1787.6 (2009):753
 Aartsma, Thijs J., and Jörg Matysik, eds. Biophysical Techniques in Photosynthesis:Volume II. Vol 26. Springer Science & Business Media, 2008.
 Myers, Jeffrey A., et al. J Phys Chem Lett 1.19 (2010):2774
 Schlau-Cohen, et al. IEEE J Sel Top Quantum Electron 18.1 (2012):283
 Schlau-Cohen, et al. Nat Chem 4.5 (2012):389
 Fuller, Franklin D., et al. Nat Chem 6.8 (2014):706
 Duan, Hong-Guang, et al. J Phys Chem B 119.36 (2015):1201
Upon absorption of a single photon rhodopsin (Rho) transitions to the activated state, Rho*, which binds the heterotrimeric transducin (Gt) inducing GDP to GTP exchange and Gt activation and dissociation. Down-stream events amplify the photon-induced signal to ultimately produce a detectable electric pulse, the visual signal that is processed by vertebrate brains. To better understand these processes imaging of native photoreceptor cells and their components using different methods is a necessity.
Imaging experiments with the atomic force microscope revealed rows of Rho dimers in disc membranes of dark-adapted mouse retina (Fotiadis et al., 2003). This controversial discovery of the early 2000 was recently confirmed by cryo-electron microscopy of cryo-sectioned mouse retina (Gunkel et al., 2015). The tomograms indicated that rows from Rho pairs (tracks) are aligned parallel to the disc incisures. Progress in camera technology has created a breakthrough for low-dose electron microscopy that will lead to advances in cryo-electron tomography. Further developments have brought the fluidic force microscopy method, which has demonstrated possibilities for single cell manipulations (Guillaume-Gentil et al., 2014).
These imaging methods, their advantages and limitations will be discussed. Further, a combination of these techniques that promises high efficiency, and possibilities to integrate further biophysical methods will be presented.
Fotiadis D, Liang Y, Filipek S, Saperstein DA, Engel A and Palczewski K (2003) Atomic-force microscopy: Rhodopsin dimers in native disc membranes. Nature 421:127-128.
Guillaume-Gentil O, Zambelli T and Vorholt JA (2014) Isolation of single mammalian cells from adherent cultures by fluidic force microscopy. Lab on a chip 14:402-414.
Gunkel M, Schoneberg J, Alkhaldi W, Irsen S, Noe F, Kaupp UB and Al-Amoudi A (2015) Higher-order architecture of rhodopsin in intact photoreceptors and its implication for phototransduction kinetics. Structure 23:628-638.
The mechanism of cellular signaling via G protein-coupled receptors (GPCRs) is not completely understood. In particular, two questions have been the focus of much attention and debate: the oligomeric status of the receptor, and the nature of its interaction with the G protein. Here, we examine those questions with the M2 muscarinic receptor and the Gi1 protein using dual-color fluorescence correlation spectroscopy (dcFCS). This technique allows for unambiguous identification of coupling behavior and can differentiate between recruitment and conformational changes.
Differently tagged receptors (GFP-M2 and mCherry-M2) or G proteins (GFP-Gαi1β1γ2 and mCherry-Gαi1β1γ2) were expressed in Sf9 cells, solubilized and purified in digitonin-cholate detergent. Both samples exhibited significant cross-correlation between the two fluorescent species, indicating the existence of oligomers (at least dimers) of both receptors and G proteins. Moreover, the co-expressed receptor-G protein complex (mCherry-M2 and GFP-Gαi1β1γ2) showed cross-correlation only in the presence of agonist (carbachol), which suggests that G proteins only couple to activated receptors. These findings were confirmed by dcFCS measurements on the same systems at the membrane of live CHO cells, in which positive cross-correlations were observed between receptors, between G proteins, and between the activated receptor and G protein. Cross-correlations were also discovered between a mutant receptor (M2D103A-mCherry) and the G protein (GFP-Gαi1β1γ2) mediated by activated wild type M2 receptors, implying functional coupling within receptor oligomers.
G protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins1. GPCR function relies on allosteric regulation of the intracellular G protein-coupling domain by the extracellular facing ligand-binding domain1. Despite recently acquired structures of active and inactive conformations of a prototypical GPCR, the β2 adrenergic receptor (β2AR) 2,3, it remains unclear how ligands regulate conformational changes in receptors. Here, we utilize 19F-fluorine NMR to directly examine the conformations and dynamics of the G protein-coupling domain of the β2AR4. These studies show that β2AR conformational plasticity is highly regulated by ligand efficacy and affinity. While NMR on population and conformational dynamics, double electron-electron resonance (DEER) spectroscopy provides detail on the distribution of states5. Taken together, the ligand both influences on-pathway states associated with β2AR activation and reveals a wealth of information regarding the influence of ligand lifetimes of specific spectroscopically detected intermediates. This “fluid” exchange between distinct intermediates stands in sharp contrast to the tight regulation of protein conformation observed for rhodopsin, and may be responsible for the complex signalling behaviour observed for many GPCRs5.
1. Christopoulos, A. & Kenakin, T. G protein-coupled receptor allosterism and complexing. Pharmacol. Rev. 54, 323–374 (2002).
2. Rasmussen, S. G. F. et al. Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature 477, 549–555 (2011).
3. Rasmussen, S. G. F. et al. Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469, 175–180 (2011).
4. Kim, T. H. et al. The role of ligands on the equilibria between functional States of a g protein-coupled receptor. J. Am. Chem. Soc. 135, 9465–9474 (2013).
5. Altenbach, C., Kusnetzow, A. K., Ernst, O. P., Hofmann, K. P. & Hubbell, W. L. High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation. Proc Natl Acad Sci USA 105, 7439–7444 (2008).
Conformational equilibria of G protein coupled receptors (GPCRs) may be intimately involved in the mechanism of intra-cellular signaling and efficacy. Here we have used double electron-electron resonance (DEER), UV-visible spectroscopy, and time-resolved EPR to quantitatively explore the structure(s) of activated rhodopsin in proteolipid nanodiscs. The results reveal that the receptor exists not as a single unique structure, but in an ensemble of conformational substates whose populations are strongly modulated by activation, by the nature of the lipid environment, and by pH. Moreover, the optically defined activated state (MIIbH+) is not a single conformation, but an equilibrium mixture of at least two substates. Thus, rhodopsin activation is not analogous to a binary switch between two defined conformations, as is often assumed. The light-activated/agonist ensemble spontaneously decays with a lifetime of ~15 minutes at 20 °C and pH 6.0. This decay corresponds in time to the formation of the functionally inactive intermediates Meta III and opsin/all-trans-retinal; for the cytoplasmic sites investigated, the structures of these states resemble that of the inactive state of the receptor. The above behavior, characteristic of rhodopsin in a lipid bilayer host, is in contrast to that in detergent solutions, where the structure of the activated receptor is strongly biased toward a single pH-independent conformation and does not spontaneously return to an inactive-like structure.
Cardiac hypertrophy, characterized by the enlargement of cardiac myocytes, occurs in response to various stressors such as increased neurohormonal factors. These neurohormonal factors, including endothelin-1 (ET-1) and phenylephrine (PE), activate G protein-coupled receptors. The endothelin and -adrenergic receptor, respectively, stimulate signaling pathways which reactivate the fetal gene program in cardiomyocytes as part of a compensatory mechanism to match increased demand. One mechanism implicated in the altered gene expression program involves Cdk9, the catalytic subunit of positive transcription elongation factor b (P-TEFb). P-TEFb activity drives elongation by releasing RNA polymerase II from a promoter-proximal paused site. It also regulates co-transcriptional mRNA processing and histone modification. The objective of our study was to determine the role of P-TEFb activity in cardiac hypertrophy. We established a primary cell culture system to model the hypertrophic response to ET-1 and PE. Rat ventricular neonatal myocytes were stained for a cardiac specific marker and cell surface area analyzed by high content microscropy. Treatment with ET-1 or PE produced an increase in cell size after 24 hours. Inclusion of the P-TEFb inhibitor DRB caused a decrease in these hypertrophic effects, validating the importance of P-TEFb in our system. Despite similar responses to ligand and DRB with respect to cell size, RT-qPCR revealed distinctive gene expression profiles mediated by the two ligands and DRB. Differential expression was observed in the genes for atrial natriuretic peptide, -myosin heavy chain and -myosin heavy chain after 24 hour treatment. Further work will explore the differences in global gene expression induced by either ligand, and their sensitivity to Cdk9 activity, using RNA-seq techniques. Uncovering the variations in gene expression mediated by different hypertrophic stimuli may lead to more specific therapies for cardiovascular disease.
The role of Gαs in G protein-coupled receptor (GPCR) signalling at the cell surface is well established. However, recent evidence has revealed the presence of Gαs on endosomes and its capacity to elicit GPCR-promoted signalling from this intracellular compartment. We recently reported an unconventional role for Gαs in the endosomal trafficking of GPCRs. Using siRNAs, we reported that Gαs knock-down specifically delayed the trafficking and degradation of internalized receptors targeted to the lysosome such as the δ-opioid receptor (DOPr) and the chemokine receptor 4 (CXCR4). This effect was specific to Gαs as the depletion of Gαi3, Gβ1 and Gγ2 did not affect degradation of these GPCRs and was also independent of the Gαs-signalling effectors adenylyl cyclase and PKA. In concordance, confocal microscopy studies indicated that Gαs depletion disrupted the transfer of these GPCRs into the intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). Interestingly, all GPCRs affected by Gαs depletion were not coupled to Gαs in their cognate signalling pathways at the plasma membrane. Our current studies aimed at identifying the molecular mechanism by which Gαs regulate this GPCR endosomal sorting step. We first examined the interaction of Gαs with the endosomal-sorting complex required for transport (ESCRT) machinery involved in GPCR sorting into ILVs. We determined that Gαs interacts directly with GASP-1 and dysbindin, two accessory sorting proteins that link a subset of GPCRs (such as DOPr) to the HRS component of the ESCRT machinery. We also recently determined that Gαs alters the ubiquitination of the ESCRT-0 components through an interaction with the E3 ligase DTX3L, which blocks the efficient sorting of CXCR4 into the degradative pathway. Our observations define a novel and general regulatory role for Gαs in the post-endocytic sorting and downregulation of GPCRs. These findings reveal that Gαs plays a role in both GPCR signalling and trafficking pathways, providing another piece in the intertwining molecular network between these processes.
Delta opioid receptors (DORs) are an involved in multiple brain functions and their agonists are currently considered as promising therapeutic alternatives for a variety of disorders including chronic pain, mood and learning troubles . Substantial evidence indicates that the duration of DOR-mediated responses is very much influenced by the trafficking route these receptors follow upon activation. Classically, DORs are considered as highly internalizing and poorly recycling receptors, but more recent evidence indicates that these trafficking patterns are also influenced by the activating ligand. Here we have characterized the post-endocytic itinerary followed by DORs after internalization by DPDPE, an agonist that supports DOR recycling. We found that once internalized, DORs travel through compartments labeled by early-endosome antigen (EEA) and SNX1 (sorting endosomes) to accumulate in Rab9/manose-6-phospate receptor (M6PRs) positive structures, corresponding to late-endosomes and multi-vesicular bodies (MVBs). Interfering with transport machinery that allows retrograde recovery of M6PRs from late endosomes to the trans-Golgi network (TGN) interfered with DOR recycling, as did blockade of TGN to membrane transport. This pathway was confirmed both in HEK293 cells and neurons, indicating that internalized DORs can return to the membrane from late endosomal compartments where they have been classically considered as committed for degradation. Blocking this itinerary induced the development of the analgesic tolerance to DPDPE in animals. This data confirms that DOR recycling from late endosomal compartments protects against the development of analgesic tolerance to DPDPE.
An important role of essential hypertension and its risks can be ascribed to the endogenous hormone angiotensin II (AngII), which regulates blood volume and vascular resistance through the angiotensin II type 1 receptor (AT1R), a member of the G protein-coupled receptor (GPCR) family. The intracellular loops (ICLs) and extracellular loops (ECLs) of the GPCR have been shown to play roles in ligand binding and receptor activation. Ligands impose distinct conformations to the receptor, therefore promoting selective downstream effector activation – a phenomenon known as biased signalling. Drugs displaying functional selectivity can act through allosteric sites to further modulate receptor responses. Our lab has shown that a peptidomimetic derived from the ECL of the prostaglandin F2α receptor (FP) acted as an allosteric modulator of FP with biased signalling properties and selective physiological actions. Therefore, we hypothesized that the ECL domains of other GPCRs can be used as putative biased-allosteric modulators. We synthesized two peptides, SC0023 and SC0024, which were derived from the AT1R’s ECL2. We found that SC0023 decreased AngII-induced ERK1/2 activation, whereas SC0024 had no affect on the AngII-induced ERK1/2 activation. Conversely, SC0024 significantly inhibited proliferation of vascular smooth muscle cells in response to AngII; however, SC0023 did not significantly affect proliferation of vascular smooth muscle cells in response to AngII. It was previously shown that these peptides mediate their effects via the AT1R, and not other GPCRs, for example, the FP receptor. Radioactive ligand binding studies with 125I-AngII concluded these peptides did not compete with the orthosteric binding site of the AT1R. Because of their receptor subtype selectivity, allosteric modulators represent a promising therapeutic intervention for the management of hypertension or other cardiovascular diseases where conventional targeting of the GPCR’s orthosteric binding site has failed.
Supported by funding from the Division of Experimental Medicine and Faculty of Medicine for S.K and CIHR for S.A.L.
One of the most important classes of cell surface receptors is G-protein coupled receptors (GPCRs) that transduce extracellular stimuli to intracellular signaling events via G-proteins. G-proteins are heterotrimeric membrane associated proteins made up of Gα subunits and Gβγ heterodimers, each consisting of multiple isoforms. Inhibition of interactions between Gβγ and specific effectors has been shown to have therapeutic potential in diseases such as heart failure and inflammation. Our lab previously identified a number of small molecules that bind to a site common to many protein-protein interactions on Gβγ subunits, via a peptide competition assay. A number of these small molecules inhibit activation of various downstream effector molecules of Gβγ. We hypothesize that the ability of various molecules to inhibit Gβγ mediated activity of these effectors is dependent on the ability of these small molecules to bind to a specific subset of amino acids important for interactions with a subset of effectors. In this way, the ability of small molecules to selectively inhibit Gβγ dependent activity of downstream effectors is dependent on the binding site of these compounds. Previously, we attempted to determine the binding sites of compounds via mutagenesis of residues that had been determined to be important for peptide binding; however, of the mutants tested, none have been able to significantly affect compound binding. Here, we validate a random mutagenesis screen in yeast to identify binding sites for Gβγ inhibitors. First we tested the ability of Gallein, a well-characterized Gβγ inhibitor, to inhibit the Gβ homolog, Ste4p, mediated mating response pathway via reporter assay. Gallein was able to inhibit α-factor induced activation of mating response with an IC50 of 4.9±0.4μM. We then determined that Gallein was able to rescue α-factor induced growth arrest with a growth rescue EC50 of 11.5±4.4μM. We also determined that a number of Gβγ inhibitors are capable of inhibiting α-factor induced growth arrest, while 12155, which binds to Gβγ and causes dissociation of Gβγ from GαGDP, was able to potentiate α-factor induced mating response with an EC50 of 3.3±0.6μM. This suggests that the binding sites of these compounds are conserved in Ste4p and that the ability of Gβγ small molecule modulators to affect the Ste4p mediated mating response pathway of S. Cerevisiae could be used to determine the binding sites of these compounds. Finally, we suggest the basis of a random Ste4p mutagenesis screen for identifying binding sites of Gβγ inhibitors.
Introduction: Recent advances in drug discovery have focused on ligand bias as a strategy to design more efficacious pharmaceutical with superior clinical efficacy and reduced side-effects. The dopamine D2 receptor is a leading receptor target that could benefit from the design of functionally selective ligands, especially for the treatment of schizophrenia, Parkinson’s disease, depression, bipolar disorder, as well as other mental health disorders. Although recent D2 drug discovery efforts have focused on β-arrestin bias, few G protein biased ligands exist for the D2 receptor, which could further our understanding of the etiology of such diseases.
Aims/Methods: Using a ligand combinatorial design approach with known ligand scaffolds such as aripiprazole and cariprazine, we synthesized hundreds of ligands and determined G protein versus β-arrestin activity using high-throughput screening using GloSensor and Tango technology conducted in parallel. Further validation of ligand bias utilized orthlogous assays such as β-arrestin and Gα(i) BRET, CRE-Luc, DiscoverX, and arrestin translocation assays.
Results: An initial screen led to the discovery of 25 novel G protein-biased ligands. These ligands ranged in their efficacy from high (90-100% quinpirole Emax), medium (50-90%) to low (<50%) at the G protein pathway. Most ligands exhibit bias factors >10 (bias factor range 3-50) with several ligands demonstrating no measureable arrestin recruitment response in any orthologous assay interrogated. Antagonist and Schild analysis of G protein-biased ligands with no arrestin efficacy revealed weak arrestin antagonism (KB >100nM), suggesting these compounds act as biased antagonists. Additional rational design has led to the discovery of D2 G protein-biased full agonists with a few ligands demonstrating super agonist activity (>100% quinpirole).
Conclusions: From our efforts, a very clear structure-activity-relationship has emerged that has guided additional rational design of G protein-biased ligands. Current progress is aimed at understanding the molecular determinants of D2 G protein-biased agonism for structure-based rational design, as well as the in vivo profile at the behavioral level.
The chemokine superfamily of ~50 small secreted proteins (8-12 kDa) and their ~20 cognate 7-transmembrane receptors (7TMRs) play essential and varied roles in the cardiovascular system. However, the promiscuity and apparent redundancy of receptor-ligand interactions of the chemokine system makes probing individual interactions and downstream effects challenging. CXCL11 is an inflammatory chemokine that binds and activates two receptors involved in cardiovascular function and development, CXCR3 and ACKR3. The exchange of the N-loop of CXCL11 with that of related chemokines, CXCL10 and CXCL12, and N-terminal truncation of CXCL11 were used to interrogate receptor function and selectivity. Function of the chimeric proteins was monitored using radio ligand binding, calcium mobilization and bioluminescence resonance energy transfer (BRET) stimulated by β-arrestin-2 recruitment. Using this strategy we were able to engineer a chimeric CXCL11 that is selective for AKCR3. With this novel protein it will be possible to isolate contributions of the CXCL11-ACKR3 interaction to cardiovascular function.
The recent proliferation of synthetic cannabinoids available to the general public has become a growing global problem. These compounds are designed to evade drug control laws and are sold as “legal highs”. In Canada, synthetic cannabinoids are controlled as “similar synthetic preparations” of cannabis if they are demonstrated to be cannabinoid receptor type 1 agonists. We implemented three cell-based assays to generate pharmacological data for five emerging synthetic cannabinoids (ESCs). We determined ESC potency and efficacy relative to a panel of well-characterized benchmark cannabinoid drugs by assessing the ability of synthetic cannabinoids to suppress cAMP levels in HEK293 cells expressing human cannabinoid receptor 1 (hCB1) and in vitro neuronal activity using two complementary techniques, Ca2+ spiking and multi-electrode arrays.
Activation of CB1 receptors inhibits cAMP levels and synaptic transmission in hippocampal neurons. Data obtained by monitoring cAMP levels indicated that benchmark cannabinoids (WIN 55,212-2, CP 55,940, JWH-018, and HU-210) reduced cAMP levels with varying levels of potency and efficacy. Furthermore, the ESCs tested with this assay were full agonists with the following rank order of potency: 5F-PB-22 ≈ AB-PINACA ≈ MAM-2201 > JWH-250 > XLR-11. In comparison, WIN 55,212-2 significantly suppressed the frequency of Ca2+ spiking in hippocampal neurons with much lower potency. Inhibition of Ca2+ spiking by WIN 55,212-2 was not enhanced by blocking receptors that interact with CB1 signaling (GABAA, GABAB, and Adenosine A1). JWH-018 (2.5 μM) was more potent in inhibiting Ca2+ spiking and was blocked by the CB1 antagonist rimonabant (5 μM). Additionally, spontaneous electrical activity of hippocampal neurons was recorded for 80 min in the presence of synthetic CB1 agonists using a multi-electrode array apparatus. All benchmarks and ESCs suppressed neural activity at 10 μM, with several compounds also significantly suppressing activity at 1 μM. Rimonabant did not suppress activity and partially reversed suppression by 5F-PB-22 and, to a lesser extent, MAM-2201.
Taken together, assessment of cAMP levels enabled detailed pharmacological assessment of potency and efficacy, while both neuronal assays demonstrated the neuro-suppressive effects of synthetic cannabinoids mediated by endogenous CB1 receptors, with MEAs providing higher sensitivity than Ca2+ spiking. This is the first report concerning the pharmacological characterization of these ESCs, and adds to the body of knowledge for these emerging drugs of abuse.
Hypertension and heart failure are major health issues and there is a need to identify more effective treatments. Regulator of G protein Signaling 2 (RGS2) is highly expressed in both heart and vascular smooth muscle and regulates Gαq signaling. RGS2-/- mice are hypertensive, show enhanced responses to vasoconstrictors and lower tolerability to pressure overload. Over-expression of RGS2 reduces cardiac hypertrophy and we have shown that pharmacologically enhanced RGS2 protein expression has functional effects on G protein signaling. Therefore, we hypothesize that enhancing RGS2 protein expression is a novel route in treating cardiovascular disease.
RGS2 is rapidly degraded through the proteasome, but the specific enzymes involved are not known. Thus, the goal of the current study was to identify the molecular machinery responsible for RGS2 protein degradation. We utilized a high-throughput siRNA screening strategy to identify genes that regulate RGS2 protein levels and identified components of a putative cullin-RING E3 ligase (CRL). siRNA knock-down of F-box 44 (FBXO44) or cullin 4B (CUL4B), but not CUL4A or CUL1, led to a significant increase in RGS2 protein levels and stability. Overexpression of FBXO44 and CUL4B decreased RGS2 protein levels. FBXO44 associates with both RGS2, CUL4B and the adaptor protein DDB1 in cells resulting in a novel E3 ligase protein complex able to degrade RGS2. Studies are now underway to characterize the molecular mechanisms of the FBXO44-RGS2 interaction.
RGS proteins are difficult drug targets due to their mode of action being through protein-protein interactions. Inhibiting a more druggable protein within the degradation pathway could be a way to increase RGS2 protein levels and function.
Structure determination of integral membrane proteins and in particular of G protein-coupled receptors has made tremendous advances in the last few years. This exciting progress required a tremendous amount of methods development: Optimization of the overproduction of GPCRs in heterologous systems; increasing the probability of crystal formation by identifying high-affinity ligands for co-crystallization and the use of the T4 lysozyme technology and other fusion approaches; advancing crystallization methods, in particular the miniaturization and automation of the lipidic cubic phase crystallization method; the development of the concept of conformational thermostabilization of GPCRs; the availability of microfocus x-ray synchrotron minibeams; and progress in NMR spectroscopy methods.
In my presentation, I will cover (i) basic aspects which govern membrane protein production (ii) use NTSR1 as an example of how we progressed from plasmid to large-scale purification, stabilization, crystallization and structure determination (iii) and discuss mechanistic aspects of how a peptide G protein-coupled receptor signals.
Structural prerequisites for G-protein activation by the neurotensin receptor. Krumm BE, White JF, Shah P, Grisshammer R. Nat Commun 2015, 6, 7895.
Structure of the agonist-bound neurotensin receptor. White JF, Noinaj N, Shibata Y, Love J, Kloss B, Xu F, Gvozdenovic-Jeremic J, Shah P, Shiloach J, Tate CG, Grisshammer R. Nature 2012, 490, 508-13.
Optimising the combination of thermostabilising mutations in the neurotensin receptor for structure determination. Shibata Y, Gvozdenovic-Jeremic J, Love J, Kloss B, White JF, Grisshammer R, Tate CG. Biochim Biophys Acta. 2013, 1828, 1293.
Structural dynamics and thermostabilization of neurotensin receptor 1. Lee S, Bhattacharya S, Tate CG, Grisshammer R, Vaidehi N. J Phys Chem B, 2015, 119, 4917.
G Protein-Coupled Receptor Kinase 2 (GRK2) and 5 (GRK5) Exhibit Selective Phosphorylation of the Neurotensin Receptor in Vitro. Inagaki S, Ghirlando R, Vishnivetskiy SA, Homan KT, White JF, Tesmer JJ, Gurevich VV, Grisshammer R. Biochemistry 2015, 54, 4320.
Modulation of the interaction between neurotensin receptor NTS1 and Gq protein by lipid. Inagaki S, Ghirlando R, White JF, Gvozdenovic-Jeremic J, Northup JK, Grisshammer R. J Mol Biol 2012, 417, 95.
Our research is funded by the NIH Intramural Research Program.
G protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by promoting the exchange of GDP for GTP on the Gα subunit. A group of receptor-independent activators of G protein signaling (AGS) proteins were identified in a yeast-based functional screen of mammalian cDNA that activated the pheromone response pathway, relying on Gβγ to do so. One of these proteins, G protein signaling modulator 3 (GPSM3, also known as AGS4 and G18), acts as a guanine nucleotide dissociation inhibitor (GDI) hindering the exchange of GDP for GTP and thereby promoting the inactive state of Gα and the active state of Gβγ. The functional role of a closely related AGS protein, LGN (also known as GPSM2), suggests that GPSM3 may also play a role in cell division.
Using vascular smooth muscle cells (VSMCs) derived from Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), the effects of serum deprivation and serum replacement were examined on GPSM3 transcript and protein levels. Transcript levels were examined using reverse transcriptase real-time quantitative polymerase chain reaction (RTq-PCR) and protein levels were examined by immunoblotting. In WKY-derived VSMCs, GPSM3 transcript levels were found to be unchanged by serum deprivation and serum replacement, while protein levels could not be detected. GPSM3 transcript and protein levels in SHR-derived VSMCs both showed a decreasing trend during serum deprivation and an increasing trend during serum replacement. Studies are ongoing to further define the potential role of GPSM3 in cell division, with focus shifting to mammalian two-hybrid methods.
Phage-displayed synthetic antibody libraries consist of billions of highly diverse members with a tailored man-made diversity in the complementarity-determining regions (CDRs) of a human antigen-binding fragment (Fab) scaffold. These libraries enable the screening of antigens under controlled in vitro conditions and thus facilitate the selection of highly specific antibodies with desired properties against diverse targets, including cell-surface antigens. The latter and particularly G-protein coupled receptors (GPCRs) are highly attractive targets, as they play fundamental roles in diverse physiological and pathological functions, and thus major drug targets. Many GPCRs exhibit significant conformational flexibility in native membranes, and solubilization with detergents for purification purposes of the receptors by themselves or their complexes with intracellular signaling proteins, increases this flexibility and associated instability, rendering them difficult for structural and functional characterization. Here we describe a strategy to generate conformation-specific antibodies targeting the active conformation of a rhodopsin-arrestin complex, directly in its native membrane. The native system maintains the stability of the physiologically relevant complex. Our model system has been the rhodopsin-arrestin complex in the rhabdomeric membranes of the visual system of the Atlantic squid, Loligo pealei. Unlike the bovine visual system, squid rhodopsin forms a high affinity long-lived complex with arrestin in a light-dependent manner in absence of any receptor phosphorylation. The target complex was reconstituted by mixing light-activated squid rhodopsin in native membrane with squid arrestin. Synthetic antibodies were selected by phage-display in five rounds of selection that included also negative selection against the non-activated form of squid rhodopsin. Following the selection process, the output phage pools were analyzed by sequencing and the most abundant Fab clones were selected and purified for further characterization. Several of the isolated Fabs were assayed for specific binding to the rhrodopsin-arrestin complex in native membrane. These Fabs displayed high binding specificity towards the active conformation of the complex, demonstrating that our strategy of targeting GPCR-transducer complexes in native membrane is amenable for rapid and efficient selection of conformation-specific antibodies. Current efforts are directed towards the crystallization of the rhodopsin-arrestin complex, using the selected conformation-specific Fabs as crystallization chaperones. High-resolution structural characterization of this complex would provide insights into the interaction of GPCRs with Arrestin using native system. The structural information may pave the way to the development of novel therapeutic strategies.
Inconsistent results obtained in the identification of oligomers of rhodopsin-like GPCRs by various techniques and in various preparations has generated much debate on the role of oligomers. Estimates of size has largely focused on oligomers of the receptor yet it is the size of the receptor coupled to the G protein that is the most relevant but remains unknown. Using the photobleaching of fusions of single-molecules of GFP, we evaluated the oligomeric size of the M2 muscarinic receptor, of the G protein and of various multimeric GFP controls. Single-molecule data recorded on a homogeneous population of oligomeric controls of a known size exhibited a laser-power-dependent distribution of photobleaching steps instead of a single number of steps. However, statistical analysis of the photobleaching histograms in terms of the binomial distribution yielded reliable estimates of the oligomeric size.
Using this approach, we examined the oligomeric size of the M2 receptor and G protein in extracts that represent different stages of the signaling cycle: in the basal state, following activation by ligands, during and after coupling of the complex. The data indicate that whereas the receptor remains as a tetramer throughout the cycle, the oligomeric size of the G protein is dynamic. G proteins exist as hexamers in the basal state, couple as tetramers and uncouple from the receptor (i.e., fully activated) as dimers. Measurements with a receptor mimic to the wild-type G protein and a constitutively active mutant also yielded dimers. The spatial feasibility of a super-complex for the di-tetrameric arrangement of the receptor and G protein was computed using molecular dynamics simulations with FRET-derived spatial constraints.
The activation state of the heterotrimeric G-protein, Gαi3, is known to regulate autophagy. Proteins that promote the inactive state of Gαi3 (AGS3 and GAIP/RGS19) promote autophagy, whereas proteins that promote the active state of Gαi3 (GIV/Girdin) inhibit the process. Importantly, the precise mechanisms whereby Gαi3 exerts effects are not well understood. We recently demonstrated that activated Gαi3 and its inhibitors RGS4 and AGS3 control intracellular JNK activity, particularly from within intracellular membrane pools. Recently, Pattingre and Levine showed that a key regulator of autophagic flux following amino acid starvation was JNK-mediated phosphorylation of Bcl2, releasing the autophagy-promoting Beclin1 protein. We thus asked whether Gαi3 might regulate the activities of JNK, Bcl-2 and Beclin1 in mammalian cells. Indeed, activated Gai3 potently inhibits JNK activity and Bcl2 mediated release of Beclin1 following amino acid starvation. In further support of this novel observation, the Gαi/o inhibitor, pertussis toxin, increases P-JNK in a dose–dependent manner and RGS4 increased P-Bcl2 and LC3II/tubulin as well as 14C labelled long lived protein degradation in HEK293. RGS4 expression deficiency impaired autophagy marker LC3II in adrenal gland. Finally, intracellular Gαi3 and RGS4 coordinately modulate Bcl-2:Beclin-1 complex formation in nutrient deprivated condition. Together these results demonstrate a role for Gαi3 and its regulator in JNK/Bcl-2 and Beclin-1 signaling in the autophagy initiation.
Chemokine receptor type 4 (CXCR4), a G protein-coupled receptor, is targeted for lysosomal degradation via a ubiquitin-dependent mechanism that involves the endosomal sorting complex required for transport (ESCRT) machinery. We have recently reported that Gαs also targets CXCR4 for lysosomal degradation; however, the molecular mechanisms by which this process occurs remain poorly understood. Here, we report a role for Gαs in ESCRT-0 ubiquitination that modulates its sorting activity. We show that Gαs co-localizes and directly interacts with domain E3 ubiquitin ligase deltex-3-like (DTX3L), which is known to inhibit the activity of Atrophin-1-Interacting Protein 4 (AIP4), an E3 ligase that mediates ESCRT-0 ubiquitination. The cellular depletion of Gαs results in the hyperubiquitination of the ESCRT-0 components hepatocyte growth factor regulated tyrosine kinase substrate (HRS) and signal-transducing adaptor molecule (STAM)-1, which blocks the efficient sorting of CXCR4 into the degradative pathway. Interestingly, this process is dependent on Gαs GTPase activity. Our data suggest a mechanism whereby Gαs, via its interaction with DTX3L, modulates CXCR4 endosomal sorting by regulating the ubiquitination status of ESCRT-0.
Until recently, G protein-coupled receptors (GPCRs) were believed to be functional only at the cell surface. However, a number of GPCRs have been demonstrated to be targeted to the nuclear membrane, including the β-adrenergic receptor, βAR. We have shown that βARs are targeted to the nuclear membrane of rat adult cardiomyocytes where they are functional with respect to cellular signalling. Since most of clinical βAR ligands can easily cross the cell membrane, monitoring the signaling properties of the different populations of βARs is crucial to understand the mechanisms of these drugs and improve their efficacies. However, the use of primary adult cardiomyocytes or isolated nuclei does not allow us to study the molecular pharmacology of nuclear βAR in a manner that combines ease of screening and a relevant cell model. For that reason, we developed two cellular models of the AC16 cardiomyocyte cell line expressing nuclear FRET cAMP or MAPK ERK1/2 biosensors. We hypothesize that nuclear signalling of βAR is distinct from that of cell membrane βARs. Using high content FRET microscopy and automated image analysis, we show that isoproterenol (100 nM) increases nuclear cAMP levels and decreases nuclear MAPK ERK1/2 activity. To assess nuclear βAR signaling, we blocked cell surface βARs with the non-permeable antagonist CGP12177A (100 nM). Isoproterenol decreased cAMP levels and increased ERK1/2 activity in the nucleus. To confirm that the increase in nuclear MAPK activity is not induced by translocation of cytoplasmic ERK1/2 activate by endosomal βARs, we inhibited nuclear import with importazole (40 µM), an importin-β inhibitor. We showed that 1h pre-incubation with 40 µM importazole blocked the nuclear increase of ERK1/2 activity produced by the protein kinase C activator phorbol 12-myristate 13-acetate (PMA). However, isoproterenol still increased nuclear ERK1/2 activity. These results suggest that isoproterenol targets a nuclear population of βARs that signals directly in the nucleus to balance signalling derived from the cell membrane. We hope these experiments will help understand the nature and scope of nuclear signalling controlled by these distinct pools of receptors and, ultimately, to translate these results into development of improved treatment for heart disease.
The delta opioid receptor (DOPr) belongs to the rhodopsin-like, G protein-coupled receptor (GPCR) family. As opposed to most GPCRs, under basal conditions DOPr is mainly localized inside the cell. Indeed, using various approaches we and others have shown that less than 5% of DOPr are expressed at the cell surface. Interestingly, the density of membrane DOPr can be increased, in vivo, by chronic pain (inflammatory and neuropathic) or by chronic activation of the mu opioid receptor. Although these observations suggest that DOPr are trafficked to the plasma membrane via the regulated secretory pathway, the underlying mechanisms are still poorly understood.
In the present study, we sought to identify novel interacting partners of DOPr involved in its trafficking. To this end, we used the proximity-dependent biotin identification (Bio ID) approach coupled to mass spectrometry. The biotin ligase Bir A* was fused to the carboxy-terminal tail of rat DOPr and transfected in NG108-15 cells. Following incubation of cells stably expressing DOPr-Bir A* with biotin, the biotinylated proteins were isolated using sepharose-streptavidin beads then identified by mass spectrometry analysis. Among the 1557 identified proteins, 193 were considered as being specific and potentially interacting with DOPr. The identified proteins belong to 21 different families (ex. cell adhesion molecules, membrane receptors, transporters, trafficking, chaperones, signalling molecules). Most interestingly, many proteins known to be involved in the regulated secretory pathway and vesicular transport have also been identified: COPI and COPII protein complexes, VAMP-3, SNAP-25, Synaptobrevin, Snx-6, MunC 18, Complexin-2, Cdc 42 effector protein 1, Rab 3, Rab 7, Rab 9, Rab 34 and Girdin. Once the interaction between DOPr and these proteins is confirmed, their role in the regulation of DOPr trafficking will be investigated in vitro and in vivo. A better knowledge of these mechanisms is important to fully understand the therapeutic potential of this important drug target.
This work is supported by the CIHR grant #MOP273137 to LG, CL, and JLP.
In the last decade, our understanding GPCR signaling has been challenged with the installment of concepts such as biased signaling and functional selectivity. The finding of new compounds able to inhibit or activate only a subset of signaling pathways activated by a specific GPCR may lead to the discovery of a new generation of therapeutics. Therefore, in order to identify subsets of signaling pathways associated with a GPCR and their involvement in different pathological states; a deeper understanding of hormones responses is still required both at the molecular and phenotypic levels. Well-known for its role in numerous pathophysiological states such as hypertension, atherosclerosis and heart failure, our attention was set upon the signaling of the peptidic hormone Angiotensin II (AngII), a powerful vasoactive agent that signals through the G-protein coupled receptor (GPCR) AT1R. Notorious to induce pleiotropic signaling upon its activation, we quantified the phenotypic and integrative response of AngII in HEK293 cells expressing AT1 receptors using cell-based and label free surface plasmon resonance (SPR) technique. In order to delineate the contribution of the pathways involved, we used various pharmacological inhibitors and siRNA-strategies to evaluate the relative implication of Gαq, Gα12/13 and βγ signaling pathways in the phenotypic responses quantified through SPR assays. Our results indicate that most of the angiotensinergic response can be suppressed using a ROCK inhibitor, suggesting the major implication of Gα12/13. This result was confirmed via the siRNA strategies revealing that Gα12 is the major player in the phenotypic response while Gα13 doesn't seem to be involved. On the other end, the used of pharmacological agent indicate a minor implication of Gαq and βγ signaling pathways in the SPR response as the pre-incubation with UBOQIC, a Gαq inhibitor, or Gallein, an βγ signaling inhibitor induced little modifications of the phenotypic response. Altogether, our results demonstrate that cell-based SPR, acting as an integrative method for measuring multiple signaling pathways, can be used to compare the phenotypic responses of different GPCR system and better understand the signaling pathways responsible of overall phenotypic responses.
Cardiac hypertrophy is a critical event in the pathogenesis of heart failure. Following insult, such as hypertension, several hypertrophic agonists, including endothelin-1 and norepinephrine are released, causing activation of G-protein coupled receptors (GPCRs) and downstream signaling events. Recently, our laboratory demonstrated that in response to activation by ET-1 or the Epac selective agonist, cTOME, hydrolysis of phosphoinositol 4-phosphate (PI4P) is induced at the Golgi apparatus in neonatal cardiac ventricular myocytes, generating diacylglycerol and inactive IP2 in a PLCε-dependent manner (Zhang et al. Cell, 2013). DAG generated from this reaction activates PKD and causes downstream hypertrophic gene expression. Our group has demonstrated that under ET-1 receptor activation conditions, this process is dependent on βɣ dimers originating from the plasma membrane (Malik et al. Mol Biol Cell, 2015). Epac is a guanine nucleotide exchange factor for Rap1, and is scaffolded with PLCε at nuclear envelope in cardiac myocytes through direct interactions with mAKAP providing a mechanism for cpTOME dependent local PLC activation and PI4P hydrolysis. Surprisingly, However; we find that Gs coupled receptors including-adrenergic receptors, relaxin receptors and adenosine receptors do not stimulate PI4P hydrolysis at the Golgi. To understand this result we investigated the role of endogenous cAMP in Golgi PI4P hydrolysis. The adenylyl cyclase activator, forskolin (10uM) stimulates PI4P hydrolysis at the Golgi membrane to a level comparable to that seen with cpTOME (25% hydrolysis). In addition a phosphodiesterase type IV selective inhibitor, Rolipram (10uM) stimulates PI4P hydrolysis to a similar degree as forskolin alone (20% hydrolysis), with addition of the non-selective phosphodiesterase inhibitor, IBMX (300uM) having little effect. Taken together, this suggests that activation of PI4P hydrolysis at the Golgi membrane can be activated by cAMP in a scaffold in which a PDE4 can degrade cAMP. This data is consistent with other reports that PDE4 inhibition can stimulate cardiac hypertrophy and that PDE4 is found scaffolded along with adenylyl cyclase (particularly isoform 5) to mAKAP. This suggests that a local pool of cAMP is required for Epac-PLC dependent PI4P hydrolysis that is not accessible to cAMP generated by adrenergic receptors under our cardiomyocyte culture conditions. Further experiments will explore mechanisms by which GPCRs generate cAMP that can access mAKAP scaffolded Epac.
Drug discovery and development of opioid ligands has largely favored highly selective agonists/antagonists for a single opioid receptor (OPr); opioid analgesics, such as morphine, are primarily selective for and activate the mu opioid receptor (MOPr). However, evidence suggests an interaction with other OPrs may be beneficial in MOPr-mediated analgesia. In particular, there is evidence that simultaneous activation of MOPr in the absence, or with inhibition, of the delta opioid receptor (DOPr) can reduce the development of morphine tolerance and dependence. Thus one strategy is to design single compounds, which bind to and concurrently activate MOPr while inhibiting DOPr, as potential therapeutics for the improved clinical management of pain. Here we report on the design, synthesis, and pharmacological evaluation of a series of compounds with a tetrahydroquinoline (THQ) core and with chemically diverse substituents that alter steric and electronic characteristics. The compounds are peptidomimetics based on key elements of opioid peptides vital for activity but that have small molecule-like features to provide for bioavailability, blood brain barrier permeability, and duration of action in vivo. Our peptidomimetics were characterized in vitro by ligand binding to provide affinity (Ki) values and for potency and relative efficacy (EC50, % stimulation compared to standard agonist) using the degree of incorporation of GTPγ35S into G proteins in membranes from cells expressing MOPr or DOPr. For example, peptidomimetic 10m showed similar binding to MOPr (Ki=0.19 nM) and DOPr (Ki=0.89 nM) and behaved as MOPr agonist (EC50=6 nM, 91% stimulation compared to DAMGO) and DOPr antagonist (antagonist affinity constant Ke=4.6 nM). In the mouse warm water tail withdrawal assay, 10m showed maximal analgesic efficacy that lasted for more than 3 hours. Data analysis and docking of 10m and other peptidomimetics into the MOPr active and DOPr inactive crystal structures have revealed structure-activity relationships to guide further chemical synthesis of improved analogues with MOPr agonist/DOPr antagonist activity. Funded by DA-03910 and the Pharmacological Sciences Training Program (NIGMS-GM007767)
Orphan G protein-coupled receptors have no known endogenous ligands and make up approximately 30% of the non-olfactory GPCRs. These understudied receptors have potential pharmacological and therapeutic relevance. Several subtypes of orphan GPCRs are expressed exclusively in the brain and/or spinal cord and may be tractable targets for the treatment of pain. Using a high-throughput β-arrestin recruitment screening platform, we discovered that a previously unstudied family of orphan GPCRs is activated by clinically relevant opioid ligands and endogenous opioid peptides. We confirmed these activities using calcium flux and PI hydrolysis assays and created preliminary structure-activity relationships (SAR) that define the pharmacological properties of this new class of opioid receptor. Our data suggest that this receptor may be a novel Gαq-coupled opioid receptor with physiological relevance.
As it is currently understood, GPCRs adopt a broad range of active and inactive conformations untill a ligand binds to it and stabilizes a subset of these conformations.
Consequently, restricting ligand conformation can hypothetically directly influence receptor conformation and subsequently biaise the involvement of the various intracellular effectors. This conformation restriction can also bring an enthropic gain upon binding, if the restricted ligand resembles more the bound conformation than the freely rotating ligand.
We therefore decided to study the effect of macrocyclization on the peptide neurotensin 8-13 (NT 8-13), which is the shortest fully active sequence of neurotensin, the endogenous ligand of NTS1.
Current studies demonstrate that activation of NTS1 is associated with antinociceptive effects1 comparable to mainstream opioids, hopefully with much less adverse effects such as dependency, desensitization, GI and respiratory side effects.
Using Ring-Closing Metathesis (RCM), we synthesized various macrocyclic analogues of NT 8-13, to reduce their conformationnal freedom. Our first goal was to mimick a beta-turn, as it has been shown that numerous peptidergic ligands for GPCRs adopt this conformation inside the binding pocket.
First-generation macrocycles were sythesized on solid phase, via RCM between two allylglycine residues.
Macrocycles affinity for NTS1 was determined using radioligand binding assay, then their efficacy to activate NTS1 was determined using BRET (Bioluminescence Resonance Energy Transfer), which monitors G protein dissociation.
Modelling of the macrocycles allowed us to compare their structure to the conformation adopted by NT 8-13 in the receptor according to the crystal structure.2
These hypotheses led to the design and synthesis of custom allylated amino acids produced from serine, proline and tyrosine. Such homemade linkers can be used to make second-generation macrocycles with improved conformation regarding the binding pocket of the receptor.
So far, macrocycles with affinities in the low micromolar range and activating the Gq pathway were identified.
1) Dobner, P CMLS 2005
2) Grisshammer, R et al. Nature 2012
The mu opioid receptor (MOPr) represents one of the most pharmacologically targeted GPCRs. Activation by orthosteric agonists such as morphine and methadone causes robust pain relief but also results in unwanted effects including respiratory depression, constipation, and addiction liability. However, these actions have different agonist efficacy requirements. Consequently an understanding of the factors governing efficacy is required. In addition, we have recently described a series of positive allosteric modulators (PAMs), exemplified by the thiazolidine BMS-986122 that control the affinity and/or efficacy of orthosteric MOPr ligands. Here we report the hexahydro-xanthene-dione, BMS-986187, a delta opioid receptor (DOPr) PAM, although structurally very distinct from BMS-986122 acts as a MOPr-PAM with increased allosteric efficacy compared to BMS-986122, but with the same probe dependence and the same mechanism of action: by allosterically disrupting Na+ binding. Furthermore, competition experiments suggest that BMS-986187 binds to the same site as BMS-986122 indicating a somewhat conserved allosteric binding site on MOPr and DOPr. Utilizing purified MOPr reconstituted into high density lipoprotein (HDL) particles we have studied binding of the MOPr state-sensitive sensor nanobody39 (Nb39) by interferometry to monitor formation of MOPr active state. Differences in orthosteric ligand efficacy were seen to correlate with different kinetics of Nb39 association and dissociation. The allosteric modulators alone enhanced the on-rates of Nb39 but differences were seen reflective of distinct allosteric efficacies. Finally, in experiments with both orthosteric and allosteric agonists we show that cooperativity between agonist and BMS-986187 is unchanged in the absence or presence of G proteins suggesting that the allosteric modulators alone are able to stabilize of an active state of MOPr. Supported by DA039997.
Recent evidence suggests that X4-2-6, a peptide derived from the 2nd transmembrane helix (TM) of chemokine (C-X-C motif) receptor 4 (CXCR4), disrupts CXCR4:α1A/B-adrenoceptor (AR) heteromerization and inhibits receptor function. Properties of peptides derived from other TM of CXCR4, however, are unknown. Here, we tested the effects of peptides derived from TM2 (X4-2-6), TM4 (X4-4-2) and TM7 (X4-7-3) of CXCR4 on receptor heteromerization and function. As assessed with proximity ligation assays, we observed that X4-2-6 and X4-7-3 reduced CXCR4:α1A-AR heteromerization in human vascular smooth muscle cells (hVSMC), but did not interfere with CXCR4:atypical chemokine receptor 3 (ACKR3) heteromerization. X4-4-2, however, reduced CXCR4:ACKR3 heteromerization, but did not affect CXCR4:α1A-AR complexes. In FACS analyses, none of the peptides affected the fluorescence signals for CXCR4, ACKR3 or α1A-AR on the cell surface of hVSMC, whereas incubation of hVSMC with receptor specific siRNAs significantly reduced the corresponding FACS signals. X4-2-6 and X4-7-3 inhibited phenylephrine induced Ca2+ fluxes and contraction of hVSMC. All peptides inhibited CXCL12 induced chemotaxis of hVSMC and freshly isolated human monocytes. Our findings point towards TM2 and TM7 of CXCR4 as possible contact sites for interactions with α1A-AR and TM4 as a contact site for ACKR3. These data further support the notion that disruption of CXCR4:α1A-AR complexes inhibits α1-AR function and suggest that all TM derived peptides that we tested function as CXCR4 inhibitors.
The metabotropic glutamate receptor 2 (mGluR2) is a member of the class C-type GPCRs activated by L-glutamate, and plays a role in normal brain function. As a presynaptic receptor found on glutamatergic neurons, mGluR2 provides negative feedback controlling glutamate release in pathways involved in learning and memory. As such, inhibition of mGluR2 activity has been proposed as a therapeutic strategy to improve cognitive function. However, identifying selective antagonists that interact at the orthosteric binding site has proven difficult due to the high degree of homology between glutamate receptor family members. Screening efforts have turned towards allosteric modulators to enable more selective modulation of different subtypes. We have identified and characterized negative allosteric modulators of mGluR2, using both functional and radioligand binding assays. Detailed kinetic and equilibrium binding studies were performed to quantify the allosteric properties of mGluR2 NAMs. Differences were observed in orthosteric ligand binding when experiments were done in the presence or absence of GTP. In the absence of GTP, the NAMs decreased the Bmax and increased the off rate of the orthosteric ligand. However, the NAMs did not impact orthosteric binding when GTP was included in the assay. Given that GPCRs are dependent on G-proteins for signaling, our work has lead us to propose that mGluR2 NAMs elicit a conformational change in the receptor that biases the receptor from coupling to the G-protein complex which essentially eliminates the ability of the receptor to respond to glutamate. Current efforts are focused to understand whether this modulation can also be observed in native tissue. This proposed mechanism may be a common feature of other allosteric GPCR modulators.
G protein-coupled receptors (GPCRs) are the largest receptor family involved in cellular signaling. The complex signaling signature of each GPCR can consist of signals dependent on one or more G proteins, β-arrestin as well as other direct partners of the receptors. To add to the complexity, ligands have been identified that only activate specific subsets of potential signaling pathways, which is called functional selectivity. It is believed that functional selectivity is based on the ability of ligands to stabilize different receptor conformations leading to the engagement of different effectors. How these conformations differ, whether they can exist independently of each other, and which interactions stabilize these conformations is still unknown.
Here, we investigate the effects of a series of mutations within the transmembrane (TM) regions of the beta-2-adrenergic receptor (β2AR) on its signaling signature. Twenty-eight mutations were introduced at 8 different residue positions, which had either been identified using Evolutionary Trace (ET) as being functionally important to receptor function or selected from visual examination of the receptor 3D structures. Mutations were selected to have different degrees of impact on signaling bias using Evolutionary Action (EA). The signaling signatures of these mutants were characterized via BRET-based biosensors and cell surface ELISA to measure multiple signaling pathways (Gs-/ Gi-activation, cAMP-levels, β-arrestin engagement, endocytosis) in response to isoproterenol (ISO). Then we clustered the mutants based on their signaling signatures deploying non-negative matrix factorization. Furthermore, using in silico mutagenesis and energy minimization we predicted changes within 4.5 Å of the mutation in the inactive (pdb: 2RH1) and the active (pdb: 4LDE) structure. The resulting 4 clusters are not only characterized by common signaling signatures but also demonstrate significant structural clustering in regards to the location of the mutation site and the predicted structural changes. Our data corroborates that the conformation of the NPxxY-motif in TM7 is critical for β-arrestin engagement since mutations affecting this region directly or indirectly display a strong decrease in β-arrestin engagement. Our data also suggests that the position of TM6 and thereby the shape of the G-protein binding pocket determines the preference between Gs- and Gi-engagement. These observations suggest that different regions of the receptor are important for the stabilization of different receptor conformations causing signaling bias. Our study may provide a first glance at the molecular determinants of signaling bias after ligand-receptor binding.
The Class Frizzled of G protein-coupled receptors consists of smoothened and Frizzled 1-10 (FZD1-10). FZDs bind and are activated by 19 mammalian WNTs, which are secreted lipoglyocproteins. For a long time, the question if FZDs indeed bind and activate heterotrimeric G proteins has been a matter of debate, despite strong evidence for an important role of G proteins in cellular WNT/FZD communication. Recent evidence further supports the idea that heterotrimeric G proteins of different groups, such as Gi/o, Gs, Gq/11 and G12/13 play a central role in FZD signalling even though we are still far away from understanding the detailed interplay between WNTs, FZDs, putative single-transmembrane spanning co-receptors and intracellular effectors.
Recent efforts in my laboratory address FZD-FZD interactions, FZD-G protein coupling, FZD-DVL binding and FZD posttranslational modifications to better understand ligand-induced dynamics in receptor complex composition and receptor-effector coupling. Systematic analysis of FZD-G protein inactive state assembly reveals interesting, previously unappreciated G protein selectivity among FZD isoforms. In addition, we uncovered a novel dynamic FZD dimerization mode, where WNT stimulation leads to dissociation of FZD dimers, a process that is important for agonist-induced signal initiation. In parallel with complex dynamics we also investigate the potential importance of posttranslational modifications of FZDs for determination of complex composition. We have identified a novel phosphorylation site conserved among all FZDs that is essential for DVL recruitment and which could provide an important switch for selection of DVL over G protein pathway signaling.
Here, I will summarize our recent efforts employing molecular and cell biology, live cell imaging and pharmacological approaches shedding light on mechanisms of FZD-mediated signal specification and initiation. Still, technical difficulties and lack of small molecule compounds targeting FZDs are limiting factors for the detailed dissection of WNT-binding, receptor activation and pathway initiation.
Neurons have an important role in retinal vascular development. Here we show that the G protein–coupled receptor (GPCR) coagulation factor II receptor-like 1 (F2rl1; also labelled protease-activated receptor 2 [PAR2]) is abundant in retinal ganglion cells and is associated with new blood vessel formation during retinal development and in ischemic retinopathy beginning with inflammatory injury; F2rl1 is induced by IL-1. After stimulation, F2rl1 in retinal ganglion cells translocates from the plasma membrane to the cell nucleus using a microtubule-dependent shuttle that requires sorting nexin 11 (Snx11). At the nucleus, F2rl1 facilitates recruitment of the transcription factor Sp1 to trigger Vegfa expression and, in turn, neovascularization. In contrast, classical plasma membrane activation of F2rl1 leads to the expression of distinct genes, including Ang1, that are involved in vessel maturation. Mutant versions of F2rl1 that prevent nuclear relocalization but not plasma membrane activation, as well as microtubule disruptors (colchicine and vinblastine) interfere with Vegfa expression but not Ang1. Distinct and complementary angiogenic factors are therefore regulated by the subcellular localization of a receptor (F2rl1) that governs angiogenesis. These findings may have implications for the selectivity of drug actions based on the subcellular distribution of their targets.
Active GPCRs (R*s) in the native membrane display a variety of conformations. We have investigated how this conformational repertoire controls coupling to G protein and arrestin. Our model receptor is rhodopsin from retinal rod cells. After fast photosensory reactions, rhodopsin sequentially adopts three so-called Meta states, which then remain in equilibrium. In the first (MIIa), an ionic constraint near the ligand is broken, in the second (MIIb), transmembrane helix 6 (TM6) tilts out of the TM bundle and in the third (MIIbH+), proton uptake leads to TM5 motion and fixation of TM6.
The TM5/6 rearrangement opens an intracellular cavity in the helix bundle. X-ray structures of native opsin apoprotein at low pH show synthetic peptides from the C-terminus of the α5 helix of Gα subunit (GαCT) bound in this cavity and forming an α-helix with C-terminal reverse turn (C-cap). Main interactions include hydrogen bonding from conserved Arg1353.50 to the C-cap and hydrophobic interactions along the inner face of TM5/TM6. To elucidate the dynamics of the binding site, we studied the interaction of GαCT peptides with rhodopsin in native disc membranes by FTIR spectroscopy. Both GαCT and the receptor protein gain α-helical structure when GαCT interacts. The interaction stabilizes the last of the Meta states (MIIH+). MD simulations suggest that in the event, a conformational substate of MIIbH+ is selected. It comprises the intracellular loop 3 (ICL3) and allows tight and precise interrogation of the binding site. This final state of interaction has properties of the state seen in X-ray. We assume that the sequential reduction of conformational space (“binding funnel”) contributes to the known speed and fidelity of signal transfer from receptor to G protein in vision.
In spite of its precision, the interaction between GαCT and the receptor cavity is flexible enough to allow reorientation of GαCΤ in the course of GDP release from the nucleotide binding site in Gα. The long rigid α5 helix acts thereby as a transmission rod between the receptor and the nucleotide site. In docking and MD simulations of GαCΤ, a clockwise rotation by 60° and translation by 1.5 Å is observed.
Arrestin employs the same cavity as the G protein as a part of its receptor interaction profile. We observed that the “finger loop” (ArrFL) from the central crest region of all four arrestins shares a consensus sequence motif (E/D)x(I/L)xxxGL) with GαCT. In co-crystals of R* with an ArrFL peptide derived from rod arrestin (Arr-1), ArrFL binds to the receptor cavity in a similar way as observed for GαCT. As confirmed by the recent X-ray structure of a R*-Arr-1 holocomplex, the interaction with TM5/6 is replaced with TM7/H8.
The receptor cavity thus serves as a universal binding site for G protein and arrestin. In the case of rhodopsin, sequential interactions with a gain in structure appear to be essential to make interactions both fast and precise. Since the sequential mechanism relies on conserved domains, it may generally underlie GPCR/ G protein coupling. In these cases it may open ways of regulation via the timing of each of the interaction steps.
Corticotropin-Releasing Factor Receptor 1 (CRFR1) is a G protein coupled receptor (GPCR) expressed widely in the brain whose antagonists have been shown to demonstrate both anxiolytic- and antidepressant-like effects. We have identified a subset of PDZ domain-containing proteins that modulate CRFR1 trafficking and signalling pathways, including ERK1/2 phosphorylation of the MAPK cascade. This prompted us to investigate the mechanism by which specific PDZ proteins may alter CRFR1-mediated ERK1/2 phosphorylation. Since it has previously been shown that β-arrestin can act as a scaffold protein to facilitate the activation and localization of MAPK signaling, we sought to determine whether PDZ proteins may also alter β-arrestin recruitment to CRFR1. In these studies, preliminary data via coimmunoprecipitation experiments in HEK293 cells suggest that a subset of PDZ proteins complex with β-arrestin2 in a CRFR1 agonist independent manner. Previous studies have shown that various PDZ proteins such as SAP97, PSD95, and MAGI proteins can regulate activation of ERK 1/2 signalling, therefore our studies show interaction with β-arrestin2 is a potential mechanism through which this occurs. Future studies with various PDZ proteins containing an assortment of protein binding domains will help specify which region of these PDZ proteins is responsible for interacting with β-arrestin2. Overall, results of these studies suggest that PDZ proteins play a critical role in regulation of β-Arrestin2 recruitment and ERK 1/2 signalling in CRFR1. A further understanding of how PDZ proteins regulate GPCRs could contribute to the design and development of new pharmacological treatments and prevention strategies for a multitude of human mental illnesses.
Receptor-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP¬2¬) is critical for physiological processes, such as neurotransmission and proliferation. Recently our laboratory identified a novel pathway in neonatal rat ventricular myocytes (NRVMs), where agonist stimulate phospholipase C (PLC)-dependent hydrolysis of the PIP¬2-precursor phosphatidylinositol 4-phosphate (PI4P) on intracellular Golgi membranes. PI4P hydrolysis by PLC leads to nuclear Protein Kinase D (PKD) activation and cardiac hypertrophy (Zhang et al., 2013). PI4P hydrolysis by PLC produces the inert inositol bisphosphate (IP2) and diacylglycerol (DAG). Our hypothesis is that PLC-dependent PI4P hydrolysis represents a general mechanism for DAG generation and PKC/PKD activation. To explore this idea we tested various GPCR agonists in multiple cell types and show that agonists stimulate golgi PI4P hydrolysis in NRVMs, Mouse Embryo Fibroblasts (MEFs), HEK293 and pancreatic ductal carcinoma (PANC-1) cells. To understand the contribution of PI4P to PLC-dependent total inositol phosphate (IP) production we depleted PI4P by two independent methods: a PI 4-kinase inhibitor, phenylarsine oxide (PAO) or expression of a Golgi specific 4-phosphatase, Sac1-K2A (Sac1); and measured total IP production. PAO treatment decreased total IP production in all cell types tested. Expression of Sac1 did not affect the levels of PIP2 at the plasma membrane or receptor-dependent calcium signals but decreased total IP production of NRVMs 76.9%±1.34; MEFs 57.6%±7.11; HEK293 25%±7.11. To our surprise, Sac1 expression did not decrease total IP production in PANC-1 cells. Depletion of PIP2, using a rapamycin-induced 5-phosphatase targeted to the plasma membrane, did not significantly affect total IP production in MEFs, HEK293 and PANC-1 cells while it decreased PIP2 labeling at the plasma membrane and abolished receptor-dependent calcium signals. These results indicate that PI4P hydrolysis is a major contribution to GPCR-stimulated total IP production. To examine the contribution of PI4P hydrolysis to DAG production in PKC/PKD activation, we measured PKD activation under conditions of either PI4P or PIP2 depletion. PAO treatment resulted in a dramatic inhibition of the ET-1-dependent global PKD activation in NRVMs and MEFs. In contrast PI4P depletion did not inhibit NT-dependent PKD activation in PANC-1 cells. Sac1-dependent depletion of PI4P in the golgi reduced ET-1-dependent global PKD activation in NRVMs and MEFs but did not change the NT-dependent PKD activation in PANC-1 cells. These results suggest that though Golgi PI4P hydrolysis occurs in multiple cell types, NRVMs and MEFs use PI4P hydrolysis as a source for DAG to promote PKD activation. In PANC-1 cells, although GPCRs stimulate Golgi PI4P hydrolysis and PI4P contributes the majority of the total IP produced, an alternative pathway is involved in PKD activation. Overall we conclude that PI4P hydrolysis at the Golgi is a major reaction stimulated by GPCRs. This mechanism allows for PLC signaling in subcellular compartments that do not contain PIP2 and may be a means for long-term DAG generation with chronic GPCR activation.
Morphine and other opioid receptor agonists are used clinically for acute and chronic pain relief and are considered to be the gold standard for pain medication. However, these opioids also have significant side effects, which, unfortunately, are also mediated via activation of opioid receptors. These side effects include tolerance, respiratory suppression, constipation, allodynia, and dependence.
In an effort to overcome these liabilities, research has focused on developing ligands with defined selectivity profiles between the various opioid receptors (μ, κ, δ), or partial agonists, or agonists used together in combination therapy. However, these diverse approaches have a single commonality in that they target the orthosteric (endogenous) agonist-binding site of the receptor. A different approach that is currently being employed with other GPCRs is the discovery and development of allosteric modulators, which bind to sites that are distinct from orthosteric binding sites, and which can have specific advantages over orthosteric ligands. These advantages include the potential for greater subtype selectivity, and, more importantly (in the case of opioid receptors), maintenance of the spatial and temporal fidelity of native signaling.
We have recently discovered the first positive allosteric modulators (PAMs) of opioid receptors, including both μ- and δ-selective PAMs. The discovery and molecular pharmacological characterization of these compounds will be described, as well as the rationale for the development of opioid PAMs as novel therapeutic agents.
The CB1 receptor represents a promising target for the treatment of several disorders including pain-related disease states. However, therapeutic applications of Δ9-tetrahydrocannabinol (THC, medical marijuana) and other CB1 orthosteric receptor agonists remain limited because of psychoactive side effects. Positive allosteric modulators (PAMs) offer an alternative approach to enhance CB1 receptor function for therapeutic gain with the promise of reduced side effects.
Here we describe the development of the novel synthetic CB1 PAM, 6-methyl-3-(2-nitro-1-(thiophen-2-yl)ethyl)-2-phenyl-1H-indole (ZCZ011), which augments the in vitro and in vivo pharmacological actions of the CB1 orthosteric agonists CP55,940 and N-arachidonoylethanolamine (AEA). ZCZ011 potentiated binding of agonists to the CB1 receptor as well as enhancing AEA-stimulated [35S]GTPγS binding in mouse brain membranes and β-arrestin recruitment and ERK phosphorylation in hCB1 cells. In the whole animal, ZCZ011 is brain penetrant, increased the potency of these orthosteric agonists in mouse behavioral assays indicative of cannabimimetic activity, including antinociception, hypothermia, catalepsy, locomotor activity and in the drug discrimination paradigm. Administration of ZCZ011 alone was devoid of activity in these assays and did not produce a conditioned place preference or aversion, but elicited CB1 receptor mediated antinociceptive effects in the chronic constriction nerve injury (CCI) model of neuropathic pain and carrageenan model of inflammatory pain. These data suggest that ZCZ011 acts as a CB1 PAM and provide the first proof of principle that CB1 PAMs offer a promising strategy to treat neuropathic and inflammatory pain with minimal or no cannabimimetic side effects and without the development of tolerance.
Pamplona et al. (2012) presented the first in vivo and in vitro evidence demonstrating that the endogenous anti-inflammatory mediator, lipoxin A4, acts as a CB1 PAM. This naturally occurring lipid enhanced both CB1 receptor binding of AEA and AEA-induced cAMP inhibition. Moreover, when given via the i.c.v. route of administration lipoxin A4 produced cannabimimetic effects (i.e., catalepsy, hypothermia, hypomotility, and antinociceptive effects in the hotplate test). Notably, systemic administration of an inhibitor of 5-lipoxygenase, the primary biosynthetic enzyme of lipoxin A4, attenuated the cataleptic effects of i.c.v administered AEA, suggesting that this endogenous lipid contributes to the behavioral actions of CB1 orthosteric agonists. More recently, the neurosteroid, pregnenolone (considered the inactive precursor of all steroid hormones) was demonstrated to modulate the effects of THC. Furthermore, the administration THC substantially increases the synthesis of pregnenolone in the brain via the activation of CB1 receptor. This reveals an previously unknown negative feedback paracrine/autocrine allosteric loop controlling CB1 function, which is mediated by pregnenalone.
The identification of the putative allosteric binding site(s) on the CB1 receptor will be a crucial step for future development of therapeutic allosteric modulators.
Previous clinical studies as well as a large number of cellular and animal behavioral studies suggest that selective activators of M1 and/or M4 subtypes of muscarinic acetylcholine receptors (mAChRs) could provide a novel approach to improving cognitive function and reducing psychotic symptoms in patients suffering from Alzheimer’s disease. In addition, mAChR agonists and have efficacy in treatment of all three major symptom clusters (positive, negative, and cognitive) in schizophrenia patients. Unfortunately, previous efforts to develop selective agonists of individual mAChR subtypes have not been successful and previous compounds have failed in development because of adverse effects due to activation of multiple mAChR subtypes. Furthermore, the relative roles of M1 and M4 in mediating the overall therapeutic effects of less selective mACh agonists are not understood. We have been highly successful in developing selective positive allosteric modulators (PAMs) of both M1 and M4 that have excellent properties for in vivo studies and potential development as drug candidates. Interestingly, selective M1 PAMs have robust efficacy in enhancing synaptic plasticity in the hippocampus and the medial prefrontal cortex (mPFC) in rodents. Furthermore, M1-selective PAMs induce robust improvements in specific domains of cognitive function in animal models that are dependent of hippocampal and mPFC function. However, M1 PAMs to not recapitulate all therapeutically relevant effects of nonselective mAChR agonists and do not have efficacy in animal models that predict antipsychotic effects. In contrast, highly selective M4 PAMs induce profound reductions in dopamine release in the nucleus accumbens and other regions that are relevant to psychosis and have robust antipsychotic-like effects in animal models. Furthermore, we found that M4 PAMs reduce transmission at cortico-striatal synapses and normalized pathological increases in cortico-striatal transmission in mice that bear mutations that give rise to Huntington’s disease. These studies provide an exciting new approach to discovery of novel highly selective activators of individual mAChR subtypes and suggest that subtype specific mAChR PAMs may provide a novel approach for treatment of multiple CNS disorders.
Double electron–electron resonance (DEER) spectroscopy is a powerful technique to obtain structural information about globular and membrane proteins, as well as protein complexes. Typically, the paramagnetic (i.e., “DEER-active”) nitroxide groups of spin labels are linked to cysteine residues of the proteins allowing for distance measurements between two spin-labelled cysteine residues. However, free rotation of bonds between the nitroxide groups and the cysteine’s α-carbon atoms complicates the deduction of precise distances within the peptide backbone. In this study, we used an intrinsically immobilized imidazoline nitroxide side chain, also referred to as V1, to minimize this effect. Using DEER spectroscopy and X-ray crystallography, we determined some of the most frequently occurring V1 rotamers on various sites of the model protein T4 lysozyme. Finally, we assessed how these rotamers are influenced by either interactions with particular neighbouring amino acid residues or by tertiary steric contacts. In conclusion, this work not only helps to better interpret the data from DEER spectroscopy experiments, but also better prepares the scientist to select more suitable sites for spin labelling.
Opioid systems in the central nervous system influence the perception of pain and contribute to mood regulation. The endogenous opioid peptides Leu- and Met- enkephalin act on the mu and delta opioid receptors (MOPr and DOPr, respectively) and function as agonists by binding to the orthosteric site of these G protein-coupled receptors (GPCRs) to promote active receptor states. Allosteric modulators bind to a site distinct from the orthosteric site on GPCRs where they can alter the binding affinity and/or efficacy of orthosteric ligands. BMS-986187 is a positive allosteric modulator of DOPr that enhances the binding affinity of orthosteric agonists at this receptor and binds with 100-fold higher affinity to DOPr compared to MOPr. However, the degree of effect of BMS-986187 on DOPr agonist affinity is not constant and is instead governed by the ligand occupying the orthosteric site, a phenomenon deemed ‘probe dependence.’ Here, we have quantified the degree to which BMS-986187 enhances the binding affinity of a wide range of DOPr orthosteric ligands, including peptides and small molecules. Binding affinities of orthosteric ligands in the absence or presence of 10μM BMS-986187 were determined by the ability of the orthosteric ligand to displace the non-selective opioid antagonist 3H-diprenorphine from human DOPr expressed in CHO cells. Na+ ions and guanine nucleotides were included in the buffer to promote inactive state(s) of DOPr. The results show that there is a 32-fold increase in binding affinity for Leu-enkephalin in the presence of BMS-986187. The non-peptide DOPr agonists TAN-67 and BW373U86 showed a similar three-fold increase in affinity as did morphine. In contrast, L-methadone, a MOPr agonist that binds poorly to DOPr, had its affinity enhanced by 170-fold in the presence of BMS-986187. The change in orthosteric ligand binding affinity induced by BMS-986187 is positively correlated with sensitivity to sodium, as previously observed with the MOPr PAM, BMS-986122. This suggests that MOPr and DOPr allosteric modulators function by a shared mechanism to promote the formation of active receptor states by disrupting Na+ binding. Understanding the mechanism and probe dependence of opioid receptor positive allosteric modulators could provide for the development of novel therapeutics to treat pain and mood disorders in the future.
The chemokine CXC-type receptor 4 (CXCR4) is a G-protein coupled receptor (GPCR) involved in many pathological processes such as HIV infection and cancer metastasis. As for other GPCRs, this receptor forms constitutive dimers and larger oligomers. CXCR4 ligands regulate the oligomers either by promoting conformational rearrangements within the pre-formed dimers or regulating assembly. Recently, a high-resolution crystallographic structures of CXCR4 dimer revealed a putative dimerisation interface involving the receptor’ helices V and VI. Using a computational screening method, we identified residues that could either stabilize or destabilize the CXCR4 dimerisation interface. We mutated these residues and tested their relevance for CXCR4 dimerisation and signalling using BRET-based approaches. As expected, BRET between wild type myc-tagged CXCR4-RLuc and HA-tagged CXCR4-YFP receptors led to a specific BRET signal, which was increased upon addition of the receptor’s natural agonist SDF-1α. Mutations predicted to stabilize the dimer, resulted in a significant decrease in the agonist promoted BRET response and in one case completely abrogated the response. However, the basal BRET signal was either not affected or even increased indicating that the stabilizing mutations maintained the receptor in a tight dimeric conformation that could not be affected by agonist stimulation. In contrast, mutations predicted to destabilize CXCR4 dimerisation resulted in significant decrease of the basal BRET signal but preservation of the agonist-promoted BRET increase. For one of these mutations, the SDF-1α-promoted BRET increase was greatly potentiated suggesting a higher flexibility of the dimer interface. Our results support the existence of a TMV-TMVI dimerisation interface for the CXCR4 and indicate that specific mutations can control the dynamics of the dimer by either restricting or facilitating inter-protomer movements. These mutant forms of the receptor provide excellent tools to better understand the role of dimerisation in CXCR4 activity.
Over 100 clinically identified rhodopsin mutations cause autosomal dominant retinitis pigmentosa (adRP), a progressive retinal degenerative disease. A majority of these mutations cause misfolding and aggregation of the receptor. It has been proposed that physical interactions between wildtype (WT) opsin and the misfolded mutant opsin may underlie the autosomal dominant phenotype. To test this possibility, we examined the interactions between WT opsin and G188R mutant opsin, a severely misfolded clinically identified adRP opsin mutant. To investigate opsin oligomerization or aggregation, we used Förster Resonance Energy Transfer (FRET) to monitor the interactions between fluorescently tagged opsins expressed in transfected HEK293 cells. In our experiments, we were able to explicitly define specific and non-specific FRET and differentiate between properly folded opsin oligomers and misfolded opsin aggregates. Wild-type opsin predominantly formed oligomers and only a minor population formed aggregates. Conversely, G188R mutant opsin predominantly formed aggregates. When wild-type opsin and G188R opsin were coexpressed in HEK293 cells, properly folded wild-type opsin did not physically interact with G188R opsin and was trafficked normally to the plasma membrane. Thus, the autosomal dominant phenotype in RP caused by misfolded opsin mutants is not predicted to arise from physical interactions between wild-type opsin and misfolded mutant opsin.
Regulator of G protein signalling 2 (RGS2) is known to play a protective role in maladaptive cardiac hypertrophyand heart failure via its ability to inhibit Gq and Gs mediated GPCR signalling. Recently, we demonstrated that RGS2 can also inhibit protein translation, which would be expected to attenuate cell growth. This novel, G protein independent inhibitory effect has been mapped to a 37 a.a. domain (RGS2eb) within RGS2 that binds to eukaryotic initiation factor 2B (eIF2B). When expressed on its own in neonatal rat cardiomyocytes, RGS2eb attenuates both protein synthesis and hypertrophy induced by Gq and Gs activating agents. The objective of this study is to further elucidate the cardioprotective role of RGS2eb by determining whether transgenic (TG) mice with cardiac specific expression of RGS2eb show resistance to the development of hypertrophy in comparison to wild-type (WT) controls. Cardiac hypertrophy was induced by transverse aortic constriction (TAC). After 4 weeks, RGS2eb TG TAC mice had significantly lower heart weight/body weight ratios compared to WT TACs. The expression of hypertrophy markers, such as alpha natriuretic peptide (ANP) and β-myosin heavy chain (MHC-β), was also significantly reduced in TG compared to WT TACs. Furthermore, heart function in TG TAC mice was maintained at sham control levels, whereas WT TACs suffered significant decreases in function. Notably, cardiomyocyte size was significantly decreased in TG compared to WT TAC mice. These results suggest that RGS2eb may be inhibiting protein synthesis in vivo, and is potentially playing an important role in protecting the heart against pathological
The complex signaling properties of G protein-coupled receptors highly is highly dependent on the structural dynamics of the these versatile signaling proteins. Biophysical methods to elucidate the structural basis of GPCR signaling has relied on a number of novel methods to stabilize receptors and signaling proteins in various conformations. In this workshop session, I will discuss currently applied protein engineering approaches to stabilize receptors for biophysical study, including X-ray crystallography, NMR spectroscopy, and EPR spectroscopy. I will also show examples that illustrate the power of combinatorial biology in stabilizing GPCRs and GPCR-signaling complexes for structural study.
Corticotropin-releasing factor receptor 1 (CRFR1) and serotonin 2A receptor (5-HT2AR) have been targeted as important G protein-coupled receptors (GPCRs) for the generation of new pharmacological treatment strategies for mental illnesses. Interestingly, both of these receptors contain class I PDZ-binding motifs on their distal carboxyl termini that are required for CRFR1-mediated enhancement of 5-HT2AR signaling: a mechanism that may underlie stress-induced anxiety and depression. The structurally homologous PDZ proteins PSD-95 and SAP97 have both been implicated in schizophrenia, and previous studies have demonstrated PSD-95 to be required for hallucinogenic and atypical anti-psychotic action via 5-HT2AR activity. Therefore, understanding PDZ protein regulation of CRFR1, 5-HT2AR, and the crosstalk therein may provide important insights for the creation of next-generation treatments for mental illness. In the current studies, we observed both redundancies and specific biases for SAP97 and PSD-95 regulation of CRFR1 and 5-HT2AR. Both SAP97 and PSD-95 are important for promoting membrane stability of CRFR1 and 5-HT2AR which may be a mechanism of antagonizing β-arrestin2 recruitment. There is no apparent effect of these proteins on Gs-coupled signaling via CRFR1, however both SAP97 and PSD-95 promote Gq-coupled inositol phosphate accumulation via 5-HT2AR. Notably, endogenous SAP97 was integral for both CRF- and 5-HT-mediated ERK1/2 phosphorylation; however PSD-95 appears to have no effect despite extensive structural homology. Neither SAP97 nor PSD-95 appear to be exclusively responsible for CRFR1-mediated enhancement of 5-HT2AR signaling. Therefore, we begin to tease out the biases of PDZ protein regulation of GPCR trafficking and signaling.
The Melanocortin Receptor Accessory Protein 2 (MRAP2) is a single transmembrane protein and like RAMPs or RTPs, it belongs to the family of GPCR accessory proteins. MRAP2 is a critical regulator of the energy homeostasis machinery and MRAP2 KO mice are severely obese. We and others identified MRAP2 as a regulator of the Melanocortin-4 receptor (MC4R) in rodents, human and zebrafish. Indeed, MRAP2 can potentiate MC4R signaling in mammals and regulate MC4R positively or negatively in the zebrafish depending on the orthologue present (MRAP2a or MRAP2b). Even though the loss of potentiation of the MC4R in MRAP2 KO mice is likely to be partly responsible for the obesity phenotype observed, we hypothesized that the activity of other important regulators of energy homeostasis would be modulated by MRAP2. In order to identify new proteins regulated by MRAP2 and better understand how MRAP2 regulates energy homeostasis, we conducted a yeast-2-hybrid screen optimized for membrane proteins. Amongst other proteins, the screen identified the Prokineticin receptor 1 (PKR1) as a new MRAP2 interacting protein. PKR1 is a GPCR that potently inhibit food intake when activated by its agonist prokineticin 2 (PK2). In addition to being the firsts non-melanocortin GPCR to be identified as a partner of MRAP2, PKR1 represents a critical but poorly understood component of the energy homeostasis machinery. To validate the yeast-2-hybrid finding we first confirmed that PKR1 and MRAP2 could form complexes in mammalian cells by co-immunoprecipitation and bi-molecular fluorescence complementation. We then found that MRAP2 causes a decrease in PKR1 expression at the cell surface as well as a significant and dose dependent inhibitory effect on PKR1 signaling. We also investigated the tissue distribution of PKR1 and MRAP2 and found that, amongst other tissues, they both express in the brain and, based on microscopic analysis, co-localize in neurons of the hypothalamus, a critical region for the control of energy intake and expenditure. Finally we validated the importance of MRAP2–mediated inhibition of PKR1 in-vivo by demonstrating that MRAP2 KO mice are more sensitive to the anorexigenic effect of PK2 than their wild type siblings. The study of the MRAP2 / PKR1 complex will not only allow a better understanding of the energy homeostatic machinery but also possibly provide us with a new target for the treatment of obesity.
Protein Arginine N-Methyltransferases (PRMTs) catalyze arginine methylation on various substrates leading to the regulation of several cellular processes, such as signal transduction, RNA processing, DNA repair and transcriptional regulation. PRMTs are classified as either Type I or Type II enzymes according to the form of methylation they catalyze. Type I enzymes produce monomethylation (MMA) and asymmetrical dimethylation (aDMA), while Type II produce MMA and symmetrical dimethylation (sDMA). In previous work, we identified PRMT5, a Type II methyltransferase, as an interacting partner of the β2-adrenergic receptor (β2AR) by a proteomic approach. Here, we confirmed the interaction between PRMT5 and the β2AR by co-immunoprecipitation assays and observed that this interaction is modulated by agonist stimulation of the receptor. Similar experiments revealed that two catalytically inactive mutants of PRMT5 (PRMT5-AAAIV and PRMT5-NN) displayed an increased interaction with the receptor, suggesting an activity-dependent role for PRMT5 on the β2AR. Using GST-pulldown assays, we identified the first intracellular loop (ICL1) of the β2AR as the major molecular determinant for this interaction. We also observed an increase of β2AR cell surface expression when HEK293 cells were treated with DsiRNAs targeting PRMT5. The opposite effect, a diminution of β2AR cell surface expression, was observed when PRMT5 was overexpressed. Agonist-induced internalization of the β2AR was unaltered by varying PRMT5 expression levels. To our knowledge, this is the first evidence of a functional interaction between a member of the PRMT family and a GPCR.
G-protein coupled receptors (GPCRs) relay signals through their canonical association with heterotrimeric G proteins composed of Gα, Gβ and Gγ. Despite the large proportion of drugs currently on the market today that target GPCRs, there remain gaps in knowledge regarding downstream signalling of their associated Gβγ dimers. Considering there are 5 Gβ and 12 Gγ isoforms, there remains a multitude of potential unique interaction partners and signalling pathways to be characterized. In our lab, we have evidence for Gβγ regulation of transcription and RNA processing, where past ChIP-chip experiments broadly identified 800+ gene promoters associated with Gβ1 and a proteomics screen uncovered 300+ potential protein interactors for Gβ1 in HEK 293 cells. We are now validating a role for Gβγ in interacting with the promoters of a subset of the genes identified, a group of miRNA transcripts that inhibit protein translation and consequently regulate cellular functions. In addition, we are investigating how Gβγ regulates processes downstream of transcription, particularly through interactions with nucleic acid binding proteins identified in our proteomic screen such as DHX9, DDX17 and HNRNP proteins that regulate RNA processing and stabilization. We hypothesize Gβγ has the ability to influence transcription and processing of miRNAs and by extension alter downstream cellular functions. We will use ChIP-seq experiments to detect DNA sequences with which Gβ associates within the genome, siRNA knock down of endogenous Gβ1 to measure changes in miRNA transcription and processing, and immunoprecipitation and siRNA knockdown against various proteins to validate and characterize their interactions with Gβ1. We hope to shed light on non-canonical Gβγ signaling and cellular miRNA regulation.
The spatial organization of G protein-coupled receptors has undergone intense scrutiny over the past decade. GPCR dimerization has been observed in many receptor systems, but the affinity, lifetime, and physiological role of dimerization is still under investigation. PIE-FCCS is a time-resolved fluorescence method that can resolve the respective population of GPCR monomers and dimers as well as their translational mobility in live cell membranes. It does this by identifying the correlated diffusion of fluorescently-tagged receptors in a confocal detection geometry. With this technique my lab has resolved the dimerization affinity of rhodopsin over a range of concentrations and extracted an effective equilibrium constant for dimerization. Here, I will present out most recent work with two other systems: rode cone opsins and dopamine receptors.
The potency and/or efficacy of GPCR agonists may be tempered by cellular deamplifying processes such as the acceleration of G protein GTPase activity by RGS proteins. Family B/R4 RGS proteins tend to undergo rapid degradation in cells, the inhibition of which can serve as a mechanism to rapidly curtail receptor signaling. The rate of proteasomal degradation of B/R4 RGS proteins via the N-end rule pathway is thought to be determined by the first few N-terminal amino acid residues, and studies have shown that amino acid substitutions within the first three N-terminal residues can profoundly affect RGS protein half-life. It follows that sequence variations in the N-terminal regions of RGS proteins could affect their function by altering protein degradation rates. We examined the stability and function of eight different naturally occurring RGS2 variants including the four initiation variants associated with its multiple AUG start sites (RGS2 FL, RGS2 t5M, RGS2 t16M, RGS2 t33M) as well as four SNPs (RGS2 M5V, RGS2 R14I, RGS2 K18N, RGS2 G23D). The four initiation variants, which differ with respect to their first few amino acid residues, exhibited different rates of degradation. RGS2 t5M showed a much briefer half-life in HEK-293 cells than the full length protein, whereas RGS2 t16M showed the greatest stability out of the four. RGS2 t33M showed a similar half-life to RGS2 FL, as did the point substitution mutant RGS2 K18N. The three other SNPs exhibited half-lives distinct from the WT protein, with RGS2 R14I and RGS2 M5V showing greater rates of degradation while RGS2 G23D showed a reduced rate. To examine the degradation pathways of these RGS2 variants, RGS2-expressing cells were treated with the proteasome inhibitor MG132. All but one of the eight constructs showed greater resultant yields as determined by immunoblotting, the exception being RGS2 G23D, which was apparently unaffected. We further compared the eight RGS2 variants for their abilities to inhibit Gq-mediated signaling by measuring their effects on 5HT-stimulated inositol phosphates accumulation in HEK-293 cells co-transfected with the 5HT2A receptor. These inhibitory effects mostly manifested as decreases in agonist potency, and there was a good correlation between the resultant agonist EC50 values when plotted against the half-life of each variant (R2 = 0.80). Taken together, these data indicate that naturally occurring variants of RGS2 differ in their cellular half-lives, and that the ability of a given variant to inhibit GPCR-stimulated Gq signaling corresponds to its cellular stability.
In other studies we examined the role of RGS2 in the stress response. As expected, the overexpression of RGS2 in NIH-3T3 cells led to a decrease in total cellular protein, reflecting its inhibitory effect on eIF2B activity. As well, RGS2 overexpression resulted in increased protein levels of the stress-activated transcription factors ATF4 and CHOP. RGS2 did not affect ATF4 or CHOP mRNA levels nor did it promote eIF2 phosphorylation. These results point to a novel mode of translational protein upregulation, as well as a previously unrecognized role of RGS2 in the cellular stress response.
Chemokine receptors are G-protein coupled receptors (GPCR) that primarily couple to the Gi/o family of Gα-proteins. Their activation triggers dissociation of the heterotrimeric G-protein, Gα and Gβγ dimer, both of which can initiate downstream signal transduction pathways critical to chemokine function. Compared to Gα, the role of Gβγ in immunology has been well studied and characterized. Key downstream effectors of Gβγ include PI3Kγ, P-Rex, PAK/PIX and mTOR that drive cell polarization and migration. Previously, our lab has characterized 12155, a small molecule that directly binds and activates βγ signaling independent of Gαi and GPCR. Neutrophils have the ability to polarize on ICAM-1 coated slides. However, treatment with 10μΜ 12155 inhibits their ability to polarize on ICAM-1. In addition, 12155 enhances intracellular cAMP levels in neutrophils in a concentration dependent manner. cAMP has been shown to play a role in chemotaxis through cAMP-dependent protein kinase (PKA). When neutrophils are treated with 1μΜ myr-PKI, which inhibits PKA, it restores their ability to polarize on ICAM-1 in the presence of 12155. PKA has been shown to regulate PI3Kγ activity via phosphorylation. To examine the role of PKA in regulating PI3Kγ, we utilized differentiated HL-60 cells stably expressing PH-AKT tagged with GFP. Polarized HL-60s show that membrane PIP3 primarily localizes in the leading edge. Treatment with 10μM 12155 alone or in the presence of 1μΜ PKI does not significantly change PIP3 levels. Furthermore, western blot analysis shows that 1μM PKI does not affect pAKT (T308) levels in the presence of 12155. This data suggests that PI3Kγ is likely not the target of PKA in regulating neutrophil polarization. In conclusion, this data show that Gβγ activation alone by 12155 inhibits polarization and increases cAMP levels. Polarization can be restored by inhibition of PKA. This indicates that both Gαi and βγ dynamically regulates cAMP levels to control cell polarity. Future experiments would explore the target of PKA that potentially plays a role in regulating cell polarization.
β-adrenergic receptors (β-ARs) modulate calcium homeostasis in cardiomyocytes. The discovery that β1- and β3-ARs are present on the nuclear membrane in these cells lead us to ask if they were involved in regulating the concentration of Ca2+ inside the nucleus ([Ca2+]n). Using a cell-permeable, photolabile caged isoproterenol analog (cIso), in conjunction with the cell-permeable Ca2+ dye Fluo-4 AM, we demonstrated that intracellular photolysis of cIso increased [Ca2+]n in adult rat cardiomyocytes. Pre-treatment with EEDQ, which irreversibly alkylates and inactivates receptors on cell surface, did not block the ability of intracellular release of Iso to increase [Ca2+]n. In contrast, photolysis of cIso failed to increase [Ca2+]n when cardiomyocytes were loaded with a caged propranolol analogue or pretreated with ryanodine. Inhibiting Gi with pertussis toxin also failed to block the ability of intracellular Iso to increase [Ca2+]n. The IP3 receptor inhibitor 2-APB had only a modest effect on the increase in [Ca2+]n. Interestingly, pretreating cardiomyocytes with either PKI(14-22) or KT5823, selective inhibitors of PKA and PKG respectively, also prevented cIso from increasing [Ca2+]n. In contrast, PKI(14-22) had no effect on the increase in [Ca2+]n produced by intracellular photolysis of a caged endothelin-1 analog. These results suggest that, in adult cardiomyocytes, nuclear β-AR activation causes an increase in [Ca2+]n that requires activation of both PKA and PKG.
Serotonin (5-hydroxytryptamine, 5-HT) type 4 and type 7 receptors (5-HT4R and 5-HT7R) regulate many physiological processes including several CNS functions. Although their expression in endothelial cells (ECs) was demonstrated before, their role in endothelial cell physiology and pathophysiology remained unclear. The results of our studies demonstrated that 5-HT4R and 5-HT7R, endogenously expressed in ECs, may promote cell migration and adhesion. Inhibition of 5-HT4R with its antagonist RS 39604 significantly suppressed EC migration in Boyden chamber migration assay and wound healing “scratch” assay. We provided the evidence that 5-HT4R-regulated EC migration may be mediated by G13 and RhoA. Importantly, inhibition of the 5-HT4R with RS 39604 reduced EC capillary tube formation in the reconstituted basement membrane, an in vitro model of angiogenesis, indicating that 5-HT4R may be involved in migration-dependent processes. Stimulation of 5-HT7R with its agonists 5-CT and AS 19 significantly increased EC migration in both Boyden chamber migration assay and wound healing “scratch” assay . Downregulation of 5HT7R using specific siRNA inhibited baseline and serotonin (5-HT)-induced EC migration. Unlike 5-HT4R-regulated migration, 5-HT7R-regulated EC migration is mediated by PKA as demonstrated by significantly reduced 5-CT- or AS 19-induced EC migration in response to pretreatment with PKA inhibitor 14-22 amide. Additionally, we showed that both 5-HT4R and 5-HT7R may be essential for EC adhesion to extracellular matrix. Our results suggest a prominent role of 5-HT4R and 5-HT7R in promoting cell migration and adhesion, and identify these receptors as potential regulators of physiological and pathophysiological processes involving cell migration and adhesion.
The adhesion G protein-coupled receptors (aGPCRs) are a large yet still mysterious family of seven-transmembrane proteins. A defining characteristic of the aGPCR family is the conserved GAIN domain, which has autoproteolytic activity and can cleave the receptors near the first transmembrane domain. Several aGPCRs, including GPR56 & BAI1, have been found to exhibit significantly increased constitutive activity when truncated to mimic GAIN domain cleavage (“deltaNT”). Recent work from other groups has suggested that the new N-terminal stalk, which is revealed by GAIN domain cleavage, can directly activate aGPCRs as a tethered agonist. We tested this hypothesis in studies on two distinct aGPCRs, GPR56 & BAI1, by engineering mutant receptors lacking the entire NT, including the stalk. These stalklesss receptors were evaluated in a battery of signaling assays and compared to full-length wild-type and cleavage-mimicking (deltaNT) forms of the two receptors. We found that the BAI1 stalkless mutant, in multiple assays, exhibited robust signaling activity, suggesting that the membrane-proximal stalk region is not necessary for BAI1 activation. For GPR56, however, the results were mixed, with the stalkless mutant exhibiting robust activity in some signaling assays but reduced activity in others. These data support a model in which the activation of certain pathways downstream of aGPCRs is stalk-dependent, whereas signaling to other pathways is stalk-independent.
Phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) is a Rho guanine-nucleotide exchange factor (RhoGEF) that regulates cell motility and is strongly associated with cancer metastasis. P-Rex1 is synergistically recruited to the cell membrane and activated by PIP3 and heterotrimeric G protein βγ (Gβγ) subunits, positioning the enzyme downstream of multiple classes of cell surface receptors that control processes such as cytoskeleton rearrangement and cell migration. P-Rex1 has thus become an attractive therapeutic target for the suppression of cancer metastasis. However, development of inhibitors against P-Rex1 is hindered by the fact that its regulatory mechanisms are poorly understood. My goal is to define the molecular basis for regulation of P-Rex1 by PIP3 and Gβγ, principally using the technique of X-ray crystallography. The resulting structures are expected to define the surfaces and residues of P-Rex1 that are important for interaction with its regulators, information that will facilitate the rational design of therapeutics that could be used to treat P-Rex1-associated cancer. Towards this goal, we have determined crystal structures of the tandem Dbl homology (DH)/pleckstrin homology (PH) domain catalytic core of P-Rex1 in complexes with its substrate small GTPases Cdc42 and Rac1. In addition, we have determined crystal structures of the independent PH domain in complex with Ins(1,3,4,5)P4, a soluble analogue of PIP3. Using site-directed mutagenesis, we have determined residues within P-Rex1 that are necessary for both PIP3 binding and P-Rex1 activation. Interestingly, mutation of these residues does not abrogate membrane binding, suggesting that PIP3 activates P-Rex1 through an allosteric mechanism rather than through recruitment to the cell membrane where substrates are located. The atomic structures coupled with biochemical and biological data have defined the key site responsible for regulation of P-Rex1 by PIP3 and reveal potential protein−protein interaction motifs that may contribute to autoinhibition by other domains within the enzyme or may serve as binding sites for regulatory proteins such as Gβγ.
Adhesion GPCRs have large extracellular regions decorated by numerous adhesion domains and a conserved GPCR Autoproteolysis Inducing (GAIN) domain that mediates self-cleavage of the receptor. Discovery of the novel GAIN domain and determination of its high-resolution crystal structures revealed the mechanism of self-cleavage and provided the great hints as to why these receptors are regulated by tethered agonists hindered within the GAIN domain. The study focuses on our recent unpublished data on further structural/functional understanding of the extracellular domains of select adhesion GPCRs that are essential for different functions in the brain such as cortex development and synapse maturation. Our studies revealed unpredicted domains in the extracellular regions, identified the conserved surfaces of these domains, and shed light on the atomic details of how these domains interact with their extracellular ligands to regulate receptor function. In addition, we engineered and determined the structures of synthetic proteins that specifically and tightly bind to the extracellular domains of adhesion GPCRs to be used as potential lead compounds for future drug discovery. We identified the downstream G-protein that couples to a few adhesion GPCR members and developed G-protein signaling assays. Functional analysis of numerous mutants identified domains and single residues essential for basal activity and for tethered peptide binding. Our structural/functional studies serve as a starting point to understand and dissect the distinct roles of extracellular and membrane domains in the functions of adhesion GPCRs.
G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin–arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotation between the amino and carboxyl domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. The rhodopsin-arrestin interaction appears to be further stabilized by electrostatic interactions between the negative charged finger loop and the positively charged TMD bundle of rhodopsin. Furthermore, the structure reveals an asymmetric binding of arrestin to rhodopsin with regard to the relative positions of the arrestin's N–C domains in respect to the membrane. In this orientation,the C-edge of arrestin, which is comprised of conserved hydrophobic residues (F197, F198, M199, F339, and L343)could either be touched or embedded in the membrane layer to facilitate the binding of arrestin to rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signaling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.
As the number of GPCR structures solved by x-ray crystallography has grown, several GPCRs have been successfully used in structure-based drug design efforts targeting their orthosteric binding site (i.e. the site occupied by the receptor’s endogenous ligand). However, the orthosteric binding site of receptors within a given family (e.g. adrenergic receptors) is often highly conserved due to the need to bind a common endogenous ligand. Thus orthosteric ligands often exhbit poor selectivity for a single receptor subtype. Allosteric modulation of GPCR signaling is an emerging therapeutic strategy with several advantages over traditional orthosteric agonism/antagonism, including improved subtype specificity. Allosteric modulators bind outside of a receptor’s orthosteric site to regions which often display more sequence variability between receptor subtypes. Here, we sought to use inactive- and active-state crystal structures of the β2-adrenergic receptor (β2AR) to identify allosteric binding pockets on the receptor and to use virtual screening in order to identify novel allosteric modulators of β2AR. We screened the ZINC database and tested a subset of the top-scoring compounds for their effects on β2AR ligand recognition and signaling. The top compound, BRAC1, displayed low affinity for β2AR (>30 uM) but was able to diminish epinephrine affinity for β2AR threefold. BRAC1 also inhibited agonist-stimulated [35S]GTPγS binding, cAMP accumulation, and arrestin recruitment. The structure-activity relationship of BRAC1 analogs (purchased or synthesized) revealed several compounds that inhibited agonist-stimulated [35S]GTPγS binding with improved potency and appeared selective for β2AR over β1AR. We also discovered several halogenated BRAC1 derivatives that worsened epinephrine affinity for β2AR by more than tenfold. The halogenated BRAC1 derivatives displayed a probe dependence that correlated with agonist efficacy: the shift in binding affinity of full agonists was greater than the affinity shift of partial agonists. These data are consistent with an allosteric mode of action by which BRAC1 and its analogs stabilize an inactive conformation of β2AR.
Arrestins are specialized adaptor proteins that bind agonist bound phosphorylated G-protein-coupled receptors (GPCRs) to regulate the activity of the largest family of cell surface receptors. Arrestin blocks access of the G-protein to the receptor and thus terminates G-protein signaling. Mammals express four arrestins. Visual arrestins (isoforms 1 and 4) are exclusively expressed in the retina, where they deactivate signaling from rhodopsin and opsins in rod and cone cells, respectively. β-arrestins (isoforms 2 and 3) are more ubiquitiously expressed and bind hundreds of GPCRs. Because GPCRs are involved in nearly all human physiology, a detailed understanding of GPCR-arrestin interaction is of tremendous importance. Best studied is the rhodopsin-arrestin interaction in the bovine visual system. In rod cells, arrestin forms a high affinity complex with the light-activated, C-terminally phosphorylated rhodopsin, which, however, is short-lived due to the eventual loss of the retinal agonist from the opsin apoprotein. The photoreceptor system of cephalopods such as squid, is remarkably similar to its mammalian counterpart, but has important differences. The squid photoreceptor system consists of a rhodopsin-Gq signaling pathway coupled to the activation of phospholipase C. The signaling from the light-activated squid rhodopsin is quenched by squid arrestin. Light-activated squid rhodopsin forms a high affinity complex with arrestin in a phosphorylation independent manner. This is in contrast with the mammalian arrestins, since they require both receptor phosphorylation and agonist bound active conformation to form high affinity complex. In vitro, light-activated squid rhodopsin-arrestin complex formed in the native membrane is stable and long lived, since the isomerized retinal is not released from squid opsin. Here we present structural, spectroscopic, thermodynamic and kinetic insights into this interaction. We find that the C-terminal tail of squid arrestin is extremely dynamic, suggesting that the classic ‘three-element interaction’ crucial in maintaining basal conformation of mammalian arrestins, is broken in this molecule. This indicates that squid arrestin adopts a conformation that is similar to the pre-activated conformations of the mammalian arrestins. Although existing in a pre-activated like conformation, squid arrestin is highly specific towards light-activated squid rhodopsin and not to its bovine counterpart. Investigation of the molecular basis of this exquisite specificity guides us to engineer arrestin specificity for different classes of receptors for structural, therapeutic and drug screening purposes.
Trace amines (TA) are biogenic amines that are by-products of the synthesis of classical neurotransmitters within the brain. TA’s exert their effect by activating a GPCR termed trace amine associated receptor 1 (TAAR1). Studies on animals have shown that TAAR1 acts as a negative regulator of dopamine transmission. Therefore TAAR1 antagonists could be beneficial in conditions of reduced dopamine transmission such as Parkinson’s disease, which arises from the loss of dopamine neurons. Unfortunately, no antagonist for TAAR1 with in vivo activity has been identified to date. Using a TAAR1 homology model, we carried out an in silico screen of 3 million commercially available compounds. The top 42 hits predicted to have the highest affinities were ordered and their pharmacological activity on TAAR1 assessed using a BRET EPAC cAMP biosensor. From the 42 compounds tested, we obtained a 38% hit rate. We focused our attention on compound 22, our lead antagonist compound that yielded an IC50 value of 22 µM for blocking TAAR1 signalling. We then tested the effects of compound 22 (LogP = 3.30) on dopamine transmission in vivo by measuring the locomotor activity of the animals. While results show that co-injection of compound 22 enhances the locomotor stimulating effects of amphetamine and cocaine, consistent with TAAR1 antagonism in vivo. However it was later found that compound 22 had an even greater effect in TAAR1-KO animals indicating compound 22 has a different primary site of action in the brain. Using the Psychoactive screening program compound 22 has been found to not significantly bind to common GPCR and transporter targets found within the brain where further screening is in progress. In summary using in silico screening we have discovered potentially novel antagonists for TAAR1. Unfortunately, the in vivo activity of one of the hits, compound 22, is not mediated by TAAR1. Therefore discovery of the target for compound 22 and further optimization could lead to new lead compounds as a novel treatment of Parkinson’s Disease.
The melanocrotin receptor accessory protein 2 (MRAP2) is an important regulator or energy homeostasis. Indeed, deletion of MRAP2 leads to severe obesity in rodents. Except for its role in potentiating the signaling of the Melanocortin-4 receptor (MC4R), the mechanisms through which MRAP2 regulates energy homeostasis are largely unknown. It is however clear that MRAP2 regulates energy homeostasis through other mechanisms since the MRAP2 KO phenotype does not perfectly recapitulate the phenotype observed in mice lacking MC4R. Additionally, MRAP2 is expressed in neurons and tissues that do not express MC4R. In this study, we identified the Prokineticin Receptor 1, a Gs and Gq coupled receptor that regulates food intake and energy expenditure as the first non-melanocortin GPCR to be regulated by MRAP2. We show that MRAP2 and PKR1 can form complexes and that MRAP2 specifically inhibits PKR1 trafficking to the plasma membrane as well as its signaling. We also demonstrate that PKR1 and MRAP2 co-localize in neurons of the arcuate nucleus and that MRAP2 inhibits PKR1 activity in-vivo since MRAP2 KO mice are hypersensitive to the anorexigenic effect of the PKR1 agonist PK2. This study not only identifies a novel partner and role of MRAP2 but also demonstrates that MRAP2 can regulate GPCRs that are not part of the melanocortin receptor family. Such new MRAP2 partners may turn out to be promising novel targets for the treatment of obesity.
Signaling mediated by hetero-trimeric G proteins at GPCRs is differentially controlled by receptor agonists. At a molecular level this is thought to occur principally via stabilization of distinct receptor conformations by individual ligands1-4. These distinct conformations, and in some instances GPCR oligimerisation, control subsequent recruitment of effector proteins2,4,5,6. Differential efficacy is thought to result from differences in ligand:receptor affinity for different effectors. Here we report that ligand-induced G protein conformations vary according to bound ligand at the calcitonin GPCR (CTR) and result in differential effects on guanine nucleotide exchange and G protein activation. Using a combination of biochemical and resonance energy transfer techniques we demonstrate ligand-dependent changes in guanosine-5'-triphosphate affinity at the ternary complex. These arise from conformational differences in the G protein hetero-trimer, rather than receptor:G protein affinity differences. Apparent affinity and on-rate measurements for G protein association to the active receptor did not correlate with ligand efficacy. This results in divergent agonist-dependent receptor-residency times for the hetero-trimeric G protein as measured by total internal reflection fluorescence microscopy and different accumulation rates for downstream second messengers. Additionally we show that upon ligand binding there is a decrease in CTR dimer:dimer interface strength, consistent with recent structural data for the β1-adrenergic receptor7 and that G protein coupling occurs in cis for the CTR. Our findings demonstrate that factors influencing efficacy extend beyond receptor conformation(s) and provide an additional role for ligand binding in G protein-dependent signaling. This understanding for an underlying mechanism of efficacy provides a molecular description for how receptor bias can arise at GPCRs and is therefore of key importance to the field.
1. Black, J. W. & Leff, P. Operational models of pharmacological agonism. Proc. R. Soc. Lond., B, Biol. Sci. 220, 141–162 (1983).
2. Mary, S. et al. Ligands and signaling proteins govern the conformational landscape explored by a G protein-coupled receptor. Proceedings of the National Academy of Sciences 109, 8304–8309 (2012).
3. Kenakin, T. Drug efficacy at G protein-coupled receptors. Annu. Rev. Pharmacol. Toxicol. 42, 349–379 (2002).
4. Deupi, X. & Kobilka, B. K. Energy landscapes as a tool to integrate GPCR structure, dynamics, and function. Physiology (Bethesda) 25, 293–303 (2010).
5. Harikumar, K. G., Pinon, D. I. & Miller, L. J. Transmembrane segment IV contributes a functionally important interface for oligomerization of the Class II G protein-coupled secretin receptor. J Biol Chem 282, 30363–30372 (2007).
6. Harikumar, K. G., Ball, A. M., Sexton, P. M. & Miller, L. J. Importance of lipid-exposed residues in transmembrane segment four for family B calcitonin receptor homo-dimerization. Regul Pept 164, 113–119 (2010).
7. Huang, J., Chen, S., Zhang, J. J. & Huang, X.-Y. Crystal structure of oligomeric β1-adrenergic G protein-coupled receptors in ligand-free basal state. Nat. Struct. Mol. Biol. 20, 419
The role of oligomers in signalling via G protein-coupled receptors (GPCRs) has been under debate. In this study, the oligomeric nature of the GPCR signalling complex comprising the M2 muscarinic receptor (M2R) and the heterotrimeric G protein Gi1 (Gαi1β1γ2) was probed using stepwise fluorescence photobleaching. A hexahistidyl-tag and a fluorophore (eGFP) were fused at the N- or the C-terminus of M2R, and inserted into Gαi1 (at position 91) or Gγ2 without perturbing the nucleotide-binding affinity of Gαi1, the formation of Gαi1β1γ2 heterotrimers or the interaction with the receptor. Purified complexes of receptors and of G proteins were immobilized on histidine-specific surfaces and their oligomeric states were evaluated by statistical analysis of the photobleaching behaviour at a single molecule level. The single-molecule data indicate that the purified receptors are tetramers and that the G proteins form hexamers even under strong reducing conditions. However, when co-purified as the signaling complex, both proteins appear as tetramers. GTP binding reduces significantly the incidence of more than two photobleaching steps on the G protein in the presence of the receptor or a mimic (mastoparan), but it has no effect on M2R. These results were corroborated with FRET measurements at the membrane of living cells and with molecular dynamic simulations, which suggests that G proteins undergo conformational changes upon binding of M2R and GTP. We propose that these conformational changes involve movement or alignment of alpha-helical domains which results in the proliferation or disruption of the oligomeric interface between the receptor and the G protein.
Neurotransmission is one of many physiological functions that are regulated by G protein-coupled receptors (GPCRs). Two important examples are the serotonin 2A receptor (5-HT2AR) and the corticotropin releasing factor receptor 1 (CRFR1). Serotonin receptors are common targets for the treatment of schizophrenia, depression, anxiety and obesity and 5-HT2AR was shown to specifically mediate anxiety responses. CRFR1 regulates stress responses associated with anxiety and depression and can also contribute to dysregulated serotonergic signaling. Previous work from our laboratory showed that CRFR1 activation leads to the sensitization of 5-HT2AR signaling as a consequence of interactions with a common set of PSD95/Disc Large/Zona Occludens (PDZ) domain-containing proteins. Therefore, the overall objective of the study is to determine the PDZ proteins responsible for CRFR1-mediated sensitization of 5-HT2AR signaling. We investigate here the MAGI subfamily of proteins which belongs to the MAGUK family. Recent studies from our laboratory clarified the roles of SAP97 and PSD95 (which are MAGUK proteins with similar structures) and found significant differences in their effects on GPCRs. Therefore, we hypothesize that the MAGI proteins will have distinct effects on the different signaling pathways despite their homologous structures. First we verified the interaction of MAGI- 2 and 3 with the receptors using co-immunoprecipitation. We showed an interaction that was dependent on the PDZ motifs of the C-tail of each receptor. We then measured the receptors surface expression and we found that both MAGI-2 and MAGI-3 can enhance the trafficking of the 5-HT2AR to the plasma membrane and do not affect CRFR1 trafficking. We also looked at the internalization of the receptors by utilizing flow cytometry. We showed that MAGI-3 blocks the internalization of CRFR1 and none of the proteins had any effect on 5HT2AR endocytosis. We examined the effects of those MAGI proteins on the receptors function. We found no effects on the CRF-stimulated cAMP formation upon over-expression or knock-down of the proteins. On the other hand, we observed an enhancement of 5-HT-stimulated IP3 formation upon over-expression of MAGI-1 and MAGI-3 but not MAGI-2. Our data suggest that MAGI proteins have distinct effects based on the receptor being regulated as well as the signaling pathway activated. This project will clarify the role of the MAGI subfamily in the regulation of GPCRs trafficking and signaling and will eventually provide us with possible candidates for the treatment of anxiety and depression.
Constitutive macropinocytosis is generally assumed to be a non-specific uptake of fluid by some cell types; its molecular mechanism remains unknown. Recently, the calcium-sensing-receptor (CaSR) has been suggested to be the mediator of this process in macrophages. Here we report that pro-inflammatory (M1) and anti-inflammatory (M2) primary human monocyte-derived macrophages differ in their ability to take up 70 kDa dextran (a hallmark of macropinocytosis): while M2 macrophages generate ≈40 macropinosomes per cell in 15 min, M1 macrophages generate less than 1 macropinosome per cell. Macropinocytosis in the M2 cells was abolished by the removal of calcium from the medium or by the CaSR antagonist NPS2143. Interestingly, however, reduced expression of the CaSR was not the cause of the unresponsiveness of the M1 cells to calcium; on the contrary, M2 cells had ≈60-fold less mRNA encoding for the receptor than M1 cells. However, the two well-established downstream players in macropinocytosis, Rac1 (a small G protein that regulates the cytoskeleton) and phosphatidylinositol 3-kinase (PI3K) were found to be 1.7-2 times more active in the M2 macrophages. Interestingly, recruitment of heterologously expressed PI3K to the plasma membrane of M1 cells using a rapamycin-inducible system was sufficient to drive macropinocytosis (≈14 macropinosomes per cell), as was heterologous constitutive activation of Rac1 (≈65 macropinosomes per cell), while overexpression of the CaSR had no effect. Strikingly, treatment of macrophages with a positive allosteric modulator of the CaSR, R568, which binds to the transmembrane region of the receptor, was sufficient to drive macropinocytosis in the M1 cells. Our findings suggest that the difference in signalling between the M1 and M2 macrophages is attributable to a step upstream of Rac1 and PI3K activation, likely the impaired ability of the CaSR in the M1 cells to be activated via the Venus flytrap domain, while remaining responsive to the classical transmembrane stimulation.
GPCRs are complex allosteric machines that associate with other receptors, G proteins and effector partners to be functional. The formation of these complexes is essential to ensure the appropriate response to extracellular signals. Many of the proteins that associate with GPCRs are first assembled in the endoplasmic reticulum (ER). What controls how and when these associations occur have yet to be determined but is likely to be of critical importance for understanding the nature of biased signalling in different cell types. The mechanisms involved in the assembly of the GPCR signalling complex are not known. A proteomic screen to identify proteins that associate with the β2-adrenergic receptor (β2-AR) identified many of components of the Endoplasmic-Reticulum-Associated protein Degradation (ERAD) quality control system, including the valosin-containing protein (VCP/P97). In addition to its role in ERAD, VCP has been implicated in multiple cellular processes.
We hypothesized that in addition to its role in the ERAD quality control system VCP may also act to establish correct protein interactions with the GPCR responsible for more than simply quality control- essentially helping to build different responders to biased ligands according to the needs of the cell at any given time. To eliminate the effect of bulk overexpression, which may overwhelm cellular regulatory machinery and alter composition of receptor signalling complexes, we utilized a tetracycline induction system to control expression of the receptor in short pulses. The β2AR tagged with a FLAG epitope and placed in an inducible vector stably expressed in HEK 293 cells where the expression level of the protein can be directly controlled. We confirmed the interaction of VCP with moderately expressed levels of FLAG-β2AR, arguing that the interaction of FLAGβ2AR and VCP is not an artifact of overexpression of an epitope tagged version of β2AR being degraded by the ERAD quality control system. Using an siRNA-based approach, we knocked down VCP and noted that levels of FLAG-β2AR were increased in cells with lower VCP levels. We next examined the effect of loss of VCP on β2AR signalling. Reduction in VCP expression altered some aspects of β2AR signalling while leaving others unchanged. Label-free experiments showed that knocking down VCP altered the global cellular response to isoproterenol stimulation. Intriguingly, the cAMP response was unchanged after VCP knock down compared with control cells. These results suggest a role for VCP in determining how the β2AR responds to ligands
Close to 50% of all pharmaceutical agents target G protein-coupled receptors (GPCRs). In many cases, patients develop a tolerance to the effects of these drugs with prolonged exposure over time. We therefore asked whether it is possible to avoid the development of tolerance to receptor inhibition using functional selectivity. To investigate, we used the GPCR CXCR4 and its ligand CXCL12 as an example. This chemokine-receptor pair regulates cell migration and stem cell homing to the bone marrow, but also promotes cancer metastasis. Therapeutic targeting of CXCR4 relies on a single unbiased antagonist, AMD3100 that inhibits all receptor signalling. However, tolerance to the effects of AMD3100 develops rapidly because receptors accumulate on the surface of cells due to inhibition of β-arrestin induced endocytosis/desensitisation. Chemokines can then bind to the overexpressed receptors and induce a response even in the presence of the drug. To explore the use of a functionally selective drug in avoiding the development of tolerance, we first demonstrate that a peptide derived from the second transmembrane helix of CXCR4 acts as a biased antagonist. The peptide, X4-2-6, potently and selectively inhibits the function of G-proteins through an allosteric mechanism but permits β-arrestin recruitment and CXCR4 endocytosis. Secondly, X4-2-6 but not AMD3100 prevents receptor accumulation on the cell surface. The peptide retains its ability to inhibit chemotaxis after prolonged exposure, while the unbiased antagonist does not. Thus, we have identified a biased antagonist of CXCR4 that inhibits the receptor over a prolonged period of time and overcomes the problem of tolerance associated with AMD3100. This demonstrates the feasibility of using functionally selective drugs as anti-GPCR therapies to circumvent drug tolerance.
In situations of chronic pain, where opiates are repeatedly administered, mu-opioid receptors undergo changes in phosphorylation and trafficking that contribute to desensitization and addiction. Alternatively, targeting the delta-Opioid Receptor (DOR), a receptor with single-use trafficking properties, could avoid these adverse consequences of traditional pain management. However, clinically, DOR agonists have lower analgesic efficacy. We hypothesize that this is due to DOR’s limited expression on the surface of nociceptive neurons. Clinically, DOR would require an exocytic signal for insertion into the membrane and treatment of chronic pain. Our goal was to elucidate the molecular mechanism retaining the intracellular pool of DOR and determine a clinically applicable method to induce its surface delivery in vivo. We have developed a model to study intracellular DOR retention using PC12 cells combined with treatment of Nerve Growth Factor (NGF). In PC12 cells, NGF stimulates DOR retention in the trans-Golgi network, resembling the phenotype observed in nociceptive neurons. For our mechanistic evaluation, we pharmacologically inhibited known NGF effectors and quantified DOR localization via fixed and live cell immunofluorescence. We assessed functional DOR on the cell surface via live-cell monitoring of cAMP activity with the EPAC FRET sensor following DOR agonist addition and quantified in vivo analgesia in mice using the Von Frey assay. Inhibition of PI3K was both required and sufficient to induce intracellular DOR retention. Activation of PI3-Kinase or inhibition of PTEN caused an increase in the 3’phorsphorylation of the phosphoinositide PI(4)P to PI(3,4)P2 which was sufficient to stimulate surface trafficking of DOR. Further, forced surface delivery of DOR induced via PTEN inhibition increased the functional surface pool of receptors. Combined administration of a PTEN inhibitor bpV(phen) and DOR agonist SNC-80 produced an increased analgesic response in a mouse model of chronic pain. Our results implicate the 3’phosphorylation of phosphoinositols as a mechanism to control the surface delivery of functional DOR for improved analgesic response following DOR-agonist administration.
The prostaglandin F2α (PGF2α) receptor (FP) is a G-protein coupled receptor (GPCR) that plays a crucial role in parturition, as it is a mediator of uterine cell contraction. We have developed a new modulator of FP, PDC113.824 (PDC) which was shown to prevent myometrial cells contraction by selectivity inhibiting PGF2α-mediated Rho/Rock signaling cascade via Gα12, while enhancing ERK1/2 and PKC signaling via Gαq. As PDC is a peptide mimic derived from the structure of the second extracellular loop (ECL2) of FP and since its effects are specific to PGF2α signaling, we hypothesize that the compound mediates its effect through its direct binding to FP. To validate this assumption we synthesized a derivative of PDC, FAT6, which while retaining the core structure of the compound, had a benzophenone (bpa) pharmacophore added to its C terminal end, allowing for crosslinking experiments to be performed. Moreover an alkyl group was added to replace the indolizidinone moiety, as alkyl groups can react and covalently attach to azido groups in presence of copper. Altogether, these modifications would allow us to covalently attach the compound to the receptor, and later covalently link this newly formed complex to a tag compound (in this case, an azido-biotin). We show that both modifications on PCD did not alter its functional activity on ERK1/2 phosphorylation. Using the angiotensin II type 1 receptor (AT1R) for protocol optimization, we designed Angiotensin-alkyl-bpa that was also shown to retain its binding and functionality on AT1R signaling. Working with Flag-AT1R-containing insect cells membranes preparations we combined crosslinking and Copper-catalyzed azide-alkyne cycloaddition (CuACC) techniques to generate a AT1R-AngII(bpa-Pra)-biotin complex. Preliminary data show that this technique can be used to isolate binding partners of small peptides and further work should be done in order to optimize the protocol for FP.
This work is funded by CIHR and March of Dimes (SAL), and a Pharmacology and Therapeutics studentship award (LN).
We report here on experiments probing the relationship between ligand binding and signaling responses in the yeast pheromone response pathway, a relatively simple, genetically well-characterized G protein coupled system with fundamental similarities to the analogous pathways in mammals. Yeast cells expressing any given level of receptors exhibit signaling responses that appear to depend directly on the number of receptors occupied by the agonist alpha-factor. However, in comparisons of cells expressing widely varying numbers of receptors, the concentration dependence of responses to alpha-factor remains remarkably constant, indicating that the response is determined by the fractional occupancy of receptors and not the absolute number of agonist-bound receptors. Furthermore, the concentration of competitive antagonist required to inhibit alpha-factor-dependent signaling is more than 10-fold higher than would be predicted based on the known affinities of agonist and antagonist. Thus, the magnitude of the response to a particular number of agonist-bound receptors is strongly affected by the presence of unoccupied or antagonist bound receptors on the same cell surface. This behavior does not appear to be due to pre-coupling of receptors to G protein or the RGS protein Sst2p. Signaling responses may, thus, be determined by a balance of positive signals by agonist-occupied receptors and inhibitory signals by unoccupied receptors, where the inhibitory signals can be diminished by occupancy of receptors by antagonist.
‘Regulators of G Protein Signaling’ (RGS proteins) are a family of proteins that negatively regulate G protein-coupled receptors (GPCRs), acting to catalyze the hydrolysis of GTP by G-alpha subunits. Some RGS proteins are endowed with other signaling features, and RGS12 is one such protein with five additional, functional domains: a PDZ domain, a phospho-tyrosine-binding domain, two tandem Ras-binding domains, and a G-alpha-binding GoLoco motif. RGS12 expression is temporo-spatially regulated in developing mouse embryos, with strong expression in somites and developing skeletal muscle. We therefore hypothesized that RGS12 is involved in coordinating signaling during myogenesis and/or skeletal muscle regeneration. We have found that RGS12 is expressed in adult tibialis anterior and is increased three days following cardiotoxin-induced injury, supporting a role in skeletal muscle regeneration. Consistent with its coordination of myogenic programming signals, RGS12 is expressed in primary myoblasts. Myoblasts isolated from mice lacking Rgs12 had an impaired ability to differentiate into myotubes compared with myoblasts isolated from wild-type mice. Given that Pax7-positive satellite cells orchestrate muscle repair, we assessed the regenerative capacity of Pax7::Cre; Rgs12-floxed mice following cardiotoxin-induced damage. Analyses revealed that eight days post-damage, mice conditionally lacking RGS12 had attenuated repair of muscle fibers as compared to wild-type mice. These data support the hypothesis that RGS12 coordinates signaling of myogenic programming. Further experiments will address whether the RGS domain—and therefore G protein signaling—is responsible for the effect of RGS12 on muscle repair.