G protein-coupled receptors (GPCRs) represent the largest class of membrane receptors in eukaryotes. Activation of GPCRs by extracellular signals leads to conformational changes that activate the associated G protein heterotrimer, composed of the α and βγ subunits. Mutations in different G protein subunits have been characterized that have been implicated in a number of different cancers and neurodevelopmental disorders. I focus on a set of ten different de novo point mutations in the Gβ1 subunit, studying the effects of these mutations in vitro, with a view toward functionally classifying them. We developed Flag-tagged siRNA-resistant versions of the mutated Gβ subunits to study them in the absence of the wildtype Gβ1 subunit. siRNA-resistance was tested to confirm that the constructs were successfully expressed.
Our previous work has shown that Gβ1 regulates M3-mAChR-mediated calcium release in HEK 293 cells; knockdown of the Gβ1 subunit leads to a significant increase in intracellular calcium release in response to carbachol treatment. To test whether expression of siRNA-resistant mutant Gβ1 could rescue the effect of WT Gβ1 knockdown, we used aequorin, an in vitro luminescence-based functional assay measuring intracellular calcium levels. Expression of the siRNA-resistant WT Gβ1 rescued the increase in calcium release caused by Gβ1 knockdown. Next, we aimed to determine whether overexpressing the different mutants could also rescue this effect. We showed that mutations K57E, D76G and I80T resulted in a loss of function, whereas K78E and K89E exhibited a dominant negative effect even in the absence of carbachol. Alternatively, mutations A11V, A92T, M101V, D118G and I269T were able to rescue the effect of Gβ1 knockdown on M3-mAChR-mediated calcium release. To further investigate this pathway, we performed co-immunoprecipitation experiments to identify which mutations physically interact with Gαq/11, the principal Gα subunit implicated in PLC activation. We did not detect interaction between Gαq/11 and Gβ1 mutants K89E, K78E and K57E.
The diversity of α,β and γ subunits allows GPCRs to modulate a wide variety of effects. Our lab has characterized an important role of Gβγ in transcription. The dimer has been found on over 700 promoters in HEK 293 cells, which led to the discovery of an interaction between Gβ1 and RNA polymerase II, stimulated by M3-mAChR activation. Determining how the different mutants affected this interaction may help explain clinical phenotypes of patients presenting the Gβ1 mutations.
How eukaryotic cells find and interact with bacteria is a fundamental question in biology. Eukaryotic phagocytes are cells that engulf and digest bacteria. These include single-celled organisms, such as amoeba, and cell types of multicellular organisms, such as macrophages. The current dogma is that phagocytic cells use at least two types of receptors for defense against invading pathogens: one for detecting and chasing pathogens via chemotaxis and another one for recognizing and eliminating them via phagocytosis. Detection and chasing is facilitated by G-protein-coupled receptors (GPCRs), whereas recognition and elimination employ pattern recognition receptors (PRRs).
Dictyostelium amoebas are stereotypical phagocytes that prey on diverse bacteria using both processes. However, as typical phagocytic receptors, such as complement receptors or Fcγ receptors, have not been found in Dictyostelium, it remains mysterious how these cells recognize bacteria. In this project, we developed a quantitative phosphoproteomic technique to discover novel signaling components. Using this approach, we discovered an orphan class C G protein-coupled receptor (GPCR) as the long sought after folic acid receptor, fAR1 (Pan et al. Dev. Cell, 2016). We show this single GPCR simultaneously recognizes the chemoattractant folic acid and the phagocytic cue lipopolysaccharide (LPS), a major component of bacterial surfaces (Pan et al. PLoS Biology, 2018) . Cells lacking fAR1 or its cognate G-proteins are defective in folic acid chemotaxis, bacterial phagocytosis and lack multiple pathways downstream of LPS, including Ras, PI 3-kinase and actin. Computational simulations combined with experiments show that responses associated with chemotaxis can also promote engulfment of particles coated with chemoattractants. Finally, the extracellular Venus-Flytrap (VFT) domain of fAR1 acts as the binding site for both folic acid and LPS. Thus, fAR1 represents a new member of the pattern recognition receptors (PRRs) and mediates signaling from both bacterial surfaces and diffusible chemoattractants to reorganize actin for chemotaxis and phagocytosis.
G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, responsible for the control of the visual sense, immune system and blood pressure, to name a few. The variety of pathological conditions they regulate makes them the ideal target for current and future drugs. Therefore, better understanding of their mechanisms of regulation is of upmost importance for the development of better and safer therapeutics. Once activated by a ligand, receptors couple to G proteins which trigger several signaling pathways, such as mitogen-activated protein kinases (MAPK). G protein signaling at the plasma membrane can be desensitized by β-arrestins. β-arrestins are endocytic and signaling adaptor proteins, respectively promoting receptor internalization and mediating MAPK pathways both at the plasma membrane and inside the cells, independently of G proteins. There are currently a limited number of pharmacological tools to dissect β-arrestins’ signaling role independent of their endocytic role in the regulation of GPCRs.
Therefore, with the use of trafficking BRET-based biosensors, we performed a high-throughput screen on a commercial library of compounds to identify new modulators of receptor trafficking. We identified trafficking molecule #21 (Traf 21) as an inhibitor of angiotensin II type 1 receptor (AT1R) internalization. We show that Traf 21 has a slight effect on β-arrestin recruitment to AP-2 in clathrin-coated pits (CCPs) but not to AT1R. Therefore, we developed a new set of BRET-based sensors and used GST pulldown to show that Traf 21 inhibits the activation of small G protein Arf6, another protein involved in the formation of CCPs. Furthermore, we found that Traf 21 also inhibits receptor-mediated ERK1/2 signaling, so we developed another set of BRET-based sensors, used GST pulldown and a Mant-GTP in vitro exchange assay to show that Traf 21 inhibits the activation of small G protein Ras, which is upstream in the MAPK pathway. However, looking at other members of this small G protein family, we found that Traf 21 does not affect the activation of Rac and Rho.
In conclusion, we have identified Traf 21 as a new endocytic inhibitor, and also characterized it as an inhibitor of small G proteins Ras and Arf6. This small molecule, we named Rasarfin, has a dual role of inhibiting proteins that are involved in both receptor signaling to ERK1/2 and receptor internalization via CCPs. Therefore, Rasarfin could be used as a pharmacological tool to further study the role of these GTPases in the internalization and signaling of other GPCRs, as well as the role of internalized receptor/β-arrestins complexes in other scaffold signaling and cellular events.
Cardiac fibroblasts are critical for the formation of a fibrotic scar to damaged areas in the heart and in cardiac remodelling during the development of heart failure. Activation of the angiotensin II (Ang II) type I receptor (AT1R) has been shown to be a critical driver of the pro-fibrotic response, initiating signalling pathways which lead to the induction of a pro-fibrotic gene expression program. While inhibition of Gβγ signalling attenuates the fibrotic response and reduces cardiac remodelling, the role of specific Gβγ subunits in regulating the AT1R-mediated fibrotic response is unknown. Furthermore, recent work has shed light on non-canonical pathways in distinct cellular compartments regulated by Gβγ. For example, our previous work demonstrated Gβ1γ interacts with and negatively regulates the activity of the transcription factor AP-1. We have also identified Gβ1γ occupancy on the promoters of more than 800 genes through ChIP-on-chip experiments. As Gβγ occupied many promoters and interacted with various transcription factors, we sought to examine whether the dimer interacted with a protein complex that binds transcription regulators and promoter regions ubiquitously. We identified RNA polymerase II (RNAPII) as a novel Gβγ interactor in primary neonatal rat cardiac fibroblasts. Co-immunoprecipitation demonstrated that activation of AT1R by its endogenous agonist angiotensin II (Ang II) led to an increased Gβγ interaction with RNAPII. We assessed the role of either Gβ1 or Gβ2 subunits on Ang II-mediated gene expression and their potential transcriptional regulatory role. While Gβ2 knockdown had minimal effects on Ang II-mediated transcriptional responses, loss of Gβ1 dysregulated the response leading to a potentiated expression of many fibrotic genes. We determined that Gβ1 was involved in the Ang II-induced interaction with RNAPII, whereas Gβ2 regulated receptor proximal signalling. Chromatin immunoprecipitation demonstrated Ang II-mediated recruitment of Gβ1to fibrotic genes, with a trend for greater recruitment towards the 3’ end. Furthermore, Gβ1 knockdown led to dysregulation of RNAPII occupancy along fibrotic genes. Taken together, our studies reveal Gβ1γ as a novel regulator of RNAPII, shedding light on the complex roles specific Gβγ dimers play in GPCR signalling.
Regulators of G-protein Signaling (RGS) proteins attenuate G-protein signaling by binding to G-proteins and accelerating GTP hydrolysis. Selective inhibition of RGS proteins may allow increased G-protein activity with better tissue specificity than global application of a GPCR agonist. A series of covalent inhibitors, the thiadiazolidinones (TDZDs), are selective for RGS4 over other RGS proteins. Previously, we have found that RGS protein flexibility is correlated with potency of covalent inhibitors when acting at a shared α4 cysteine. We hypothesized that differences in a salt-bridges between the α4 and α5 helices in RGS proteins are responsible for differences in flexibility and potency among isoforms.
RGS4 and RGS8 share a salt bridge forming residue on the α4 helix (D90 and E84 respectively) while RGS19 lacks a charged residue (L118). When RGS19 was mutated to induce formation of an α4-α5 salt bridge (L118D), the protein showed a 7 °C increase in stability compared to WT, as measured by DSF. Conversely, a mutation removing this salt bridge in RGS8 (E84L) caused an 8 °C reduction in stability. Flexibility of mutant proteins was evaluated by hydrogen-deuterium exchange (HDX). The helix surrounding the mutation site was found to be protected from deuterium exchange on addition of a salt-bridge (RGS19 L118D), and have higher exchange on removal of a salt bridge (RGS4 D90L and RGS8 E84L). In addition, changes in the α4-α5 salt bridge affected potency of inhibition by TDZD inhibitor CCG-50014. RGS19 L118D had reduced potency of inhibition compared to WT RGS19, while RGS4 D90L and RGS8 E85L mutations increased potency of inhibition compared to WT proteins. These results show that differences in the α4-α5 salt bridge are responsible for flexibility differences among RGS isoforms, and demonstrate a causal relationship between RGS flexibility and potency of TDZD inhibitors.
Huntington’s Disease (HD) is an inherited autosomal dominant neurodegenerative disorder that leads to motor and cognitive impairment and ultimately death. Despite the devastating outcomes, there are currently no available disease modifying treatments for HD patients. Inhibition of postsynaptic metabotropic glutamate receptor 5 (mGluR5) signalling using the negative allosteric modulator, CTEP, improved HD symptoms in zQ175 knock-in mouse model of HD. However, it remains unclear whether reducing glutamate release into the synaptic cleft could provide the same or additional beneficial health outcomes in HD. Here, we are examining whether the activation of metabotropic glutamate receptor 2 and 3 (mGluR2/3), which inhibits presynaptic glutamate release, can delay/stop HD progression and pathology in zQ175 mice. We will employ LY379268 as a potent, selective and systematically active mGluR2/3 agonist, which has been shown to be effective in animal models of stroke, epilepsy and schizophrenia. More importantly, its human version is found to be safe and well tolerated in a phase-2 clinical trial in schizophrenic patients. Prior to treatment, 12-month old heterozygous and homozygous zQ175 knock-in mice exhibited significant defects in their motor, cognitive and locomotor activity, compared to age-matched wild-type mice. We then treated mice with either saline or LY379268 (3mg/kg/day) delivered via implanted osmotic pumps. After only one week of treatment, LY379268-treated heterozygous and homozygous mice showed no significant improvement in their grip strength, locomotor activity and rotarod performance in comparison to saline-treated mice of the same genotype. Mice will be tested again following one month, two months and three months of LY379268 treatment to explore whether longer exposure to the drug will result in significant improvement in motor and cognitive defects. This project will provide evidence on whether targeting mGluR2/3 to reduce glutamate release could be a viable strategy for treating HD patients.
Rationale: The heterotrimeric protein Go, whose a subunit is encoded by GNAO1, regulates ion channel function, neurotransmitter release, and neurite outgrowth. Mutations in GNAO1 have been identified in children with either epileptic encephalopathy (EIEE17) or neurodevelopment disorder with involuntary movements (NEDIM). The mechanism underlying the complex clinical spectrum of these GNAO1 encephalopathies is poorly understood. Previously, we discovered a genotype-phenotype correlation on patients’ mutations with an in vitro functional assay. De novo GNAO1 mutations have both GOF and LOF biochemical function with the former associated with seizures and the latter with movement disorder. We also reported a Gnao1 GOF knock-in mutant (Gnao1G184S/+) with a mild seizure phenotype in C57Bl/6J mice. In the current study, we assessed behavioral characteristics and electrophysiology properties and morphological changes in the cerebellum of Gnao1 mutant mouse models to explore the pathophysiology of GNAO1-associated movement disorders and to further validate our model associating GNAO1 GOF mutations with movement disorder.
Methods: Gnao1G184S/+ (GOF) and Gnao1-/+ (HetKO) animals of both sexes (age 8 to 14 weeks) were analyzed. Spontaneous inhibitory (sIPSCs) in Purkinje cells in cerebellar slices were measured using whole cell patch-clamp recording techniques. Morphology was assessed with Nissl staining. Motor capabilities were assessed using a battery of behavioral tests.
Results: Compared to Gnao1 WT mice, GOF mice showed a number of behavioral abnormalities related to movement including open field, rotarod, stride length, paw angle variability, and grip strength. HetKO mice were relatively normal in motor functions. Cerebellar slices from GOF mice showed mildly reduced sIPSC frequency. Cerebella from GOF mice also had reduced lobule number but molecular layer thickness was unchanged.
Conclusions: The Gnao1 GOF mutant mice, as with human GNAO1 GOF patients show a pronounced movement disorder. This may be related to altered inhibitory signaling in the cerebellum.
Funding: Supported by an American Epilepsy Association Predoctoral Fellowship (to H.F.)
Cardiac hypertrophy is a leading cause of morbidity and mortality in the USA. Previously, our laboratory identified a new prohypertrophic pathway where PLCe, scaffolded to mAKAPβ at the nuclear envelope, hydrolyzes phosphatidylinositol 4-phosphate (PI4P) at the Golgi. This pathway is activated by multiple upstream signals including the Epac-selective cAMP analogue, cpTOME. However, stimulation of β-adrenergic receptors (βARs) with Isoproterenol does not activate this pathway despite strongly raising cAMP. Irannejad et al2 demonstrated that β1-adrenergic receptors are present in the Golgi membrane of Hela cells and can generate cAMP there. Boivin et al3 demonstrated that β1ARs localize to the perinuclear region of adult cardiomyocytes. To determine if internal βARs can stimulate PI4P hydrolysis, we treated NRVMs with the membrane permeable βAR agonist, dobutamine. In contrast to the membrane impermeable Iso, dobutamine induced rapid and sustained PI4P hydrolysis that was blocked by the cell permeable β-adrenergic antagonist, metoprolol. The cell impermeable βAR antagonist, sotalol did not block dobutamine effects, suggesting that a pool of internal β-adrenergic receptors can induce PI4P hydrolysis. Over-expression of the RA1 domain of PLCε, which competes PLCε away from the mAKAPβ scaffold, or knockdown of PLCε also inhibited dobutamine stimulation of PI4P hydrolysis. Norepinephrine (NE), a physiological agonist of βARs, stimulated PI4P hydrolysis at the Golgi in a metoprolol sensitive manner. NE-mediated PI4P hydrolysis was abrogated by corticosterone, an inhibitor of the cation transporter Oct3. Oct3 has been recently shown to be required for epinephrine-mediated β1-AR stimulation at the Golgi envelope in HELA cells2. Taken together, these data suggest that βAR stimulation in internal membranes, potentially at the Golgi apparatus, can stimulate PI4P hydrolysis. These observations are evidence for a novel mechanism for PLC activation through an internal GPCR which may be responsible for cardiac hypertrophy and heart failure. Targeting internal β-adrenergic receptors may allow for the development of selective therapies which can treat heart failure with little to no effect on other cardiac functions.
Several membrane-mimetic systems have been developed previously to reconstitute membrane proteins, including G protein-coupled receptors (GPCRs), into model lipid bilayers. We have applied DNA nanotechnology to develop DNA-encircled bilayers (DEBs), which are composed of multiple copies of an alkylated oligonucleotide hybridized to a single-stranded DNA minicircle. The alkyl chains are designed to point towards the inside of the toroidal DNA ring. Added phospholipids are then stabilized at the hydrophobic rim of the DNA toroid to create a bilayer. The inherent programmability of DNA nanostructures allows the precise control of disk size, and facilitates site-specific functionalization on the ring structure. In preliminary studies to reconstitute GPCRs into DEBs, we have labeled the N-terminal tail of purified human C-C chemokine receptor 5 (CCR5) with an oligonucleotide, which is complementary to an oligonucleotide designed to protrude from the DNA ring. We demonstrate that oligo-labeled CCR5 can be captured on a chip surface studded with complementary oligos. Hybridization of the oligonucleotides on the CCR5 with an oligo-labeled DEB should aid with the insertion of CCR5 into the bilayer, while allowing the control of its orientation. We plan to use the DEBs to conduct single-molecule ligand-binding and pharmacological studies of CCR5 and other GPCRs, as well as using DEBS as cryoEM scaffolds.
Epileptic encephalopathy (EIEE17) and neurodevelopmental delay with involuntary movements (NEDIM) have both observed in patients with de novo mutations in GNAO1. Some mutations result in a gain of function of the encoded protein, Gαo, an adenylyl-cyclase-inhibitory G-protein highly expressed within the brain, where it mediates the production of cAMP. We used a CRISPR produced mouse model with a patient derived mutation, Gnao1+/G203R(GOF) to understand how GNAO1 haplodeficiency is implicated in NEDIM and EIEE17. Existing studies have shown a phenotype- genotype correlation linking LOF mutations with epilepsy and GOF mutations with movement disorders in children and infants with GNAO1mutations. However, children with de novo mutations in Gnao1+/G203R display both epilepsy and movement disorders, including dystonia characterized by sustained or intermittent muscle contractions. In basal conditions Gnao1+/G203R mice displayed significant deficits in behavioral tests such as the open field, rotarod, digigait and grip strength, but no abnormal postural differences were observed. After administration of oxotremorine, a cholinergic agonist, Gnao1+/G203R mice displayed increased abnormal postures and movements compared to wild type mice. We also report that Gnao1+/G203R have greater sensitivity to Pentylenetrazol (PTZ). Therefore, oxotremorine-treated Gnao1+/G203R mice provide a model to study GNAO1 associated disorders. Future studies will explore various treatment regiments aimed at alleviating movement abnormalities in the animal model.
The development of “safer” opioids is difficult owing to the fact that the undesirable effects of opioid analgesics are all on-target effects at the m opioid receptor. Our laboratory has pioneered an approach to biasing GPCR signalling with small molecules that bind directly to the Gβγ subunits to selectively modify signals downstream of GPCRs. Two prototype molecules, gallein and M119 have previously been shown to potentiate the analgesic potency of morphine, without potentiating side effects typically associated with opioid use including acute tolerance, respiratory depression, constipation, hyperlocomotion, reward preference or physical dependence [1-3].
To identify novel small molecule inhibitors of Gβγ with more drug-like potential we screened small-molecule libraries of over 170, 000 compounds comprised of diversity sets from Maybridge and Chemdiv. One lead, compound 67, inhibits in a primary Alphascreen assay with 5 μM potency and inhibits and in vitro assay of Gβγ-stimulated PLC activity with similar potency. Compound 67 also potentiates the analgesic potency of morphine in the warm water tail withdrawal (WWDW) model of analgesia. We have undertaken medicinal chemistry efforts to improve the pharmacological and physicochemical properties of compound 67 to improve solubility and potency. The most notable improvement made to compound 67 is an considerable increase in solubility. 3 derivatives have been synthesized with improved clogP (2.8, 3.1, 3.2 vs. 4.5 for compound 67) while still maintaining potency higher than 10 μM. 2 of these compounds showed efficacy in potentiating the analgesic effect of morphine in the WWDW assay.
We are further developing compound 67, as well as a number of cell based assays to elucidate the mechanism by which Gβγ inhibitors potentiate the morphine analgesia.
1. Hoot, M.R., et al., Inhibition of Gβγ-subunit signaling potentiates morphine-induced antinociception but not respiratory depression, constipation, locomotion, and reward. Behavioural Pharmacology, 2013. 24(2): p. 144-152.
2. Bianchi, E., et al., Supraspinal Gbetagamma-dependent stimulation of PLCbeta originating from G inhibitory protein-mu opioid receptor-coupling is necessary for morphine induced acute hyperalgesia. J Neurochem, 2009. 111(1): p. 171-80.
3. Mathews, J.L., A.V. Smrcka, and J.M. Bidlack, A novel Gbetagamma-subunit inhibitor selectively modulates mu-opioid-dependent antinociception and attenuates acute morphine-induced antinociceptive tolerance and dependence. J Neurosci, 2008. 28(47): p. 12183-9.
The Angiotensin II (AngII) type I receptor (AT1R) is a member of the G protein-coupled receptor (GPCR) superfamily. AT1R activation promotes the recruitment of several signaling effectors including G proteins and β-arrestins (β-arrs). β-arrs are adaptor proteins that promote GPCR desensitization and signaling. Although it has been shown that β-arrs form complexes with most GPCRs, their specific interaction and binding mode to receptors upon different ligands interaction, including for AT1R, remains elusive. Therefore, we aimed to determine the binding modality of β-arrs onto AT1R, using a bioorthogonal labeling approach and site-specific incorporation of photo-reactive unnatural amino acids (UAAs) into the receptor. We incorporated the p-azido-L-phenylalanine (azF), a highly reactive UAA that crosslinks with nearby C-H bonds upon UV light exposure, into AT1R-RlucII. Photo-reactive AT1R mutants were generated in the extracellular loops (ECLs), transmembrane domains (TMDs), intracellular loops (ICLs) and C-terminus (C-term) region of the receptor. Our data show site-specific incorporation of azF into AT1R and the photo-reactive AT1R mutants expressed in HEK293 cells are functionally active, as revealed by receptor-mediated ERK activation and intracellular calcium production following AngII stimulation. We also show that the azF-labeled AT1R mutants recruit β-arr upon AngII stimulation, and that they covalently bind β-arr1 following AngII stimulation and UV exposure. Our analysis of photo-reactive AT1R mutants reveals that β-arrs interact with residues within the ICLs and C-term region of the receptor. These studies will allow understanding how β-arrs bind with AT1R and how these interactions affect β-arr-dependent signaling pathways. Such approach can also be used with other GPCRs or ligands to shed light on the structural dynamics of ligand-GPCR-β-arr complex formation.
G protein-coupled receptor (GPCR) function within the cell is organized by location and time. This organization is maintained by the coordinated action of many cellular proteins which mediate and regulate GPCR activity in a spatiotemporally precise manner. Intriguingly, stimulating a GPCR with different ligands—such as partial, biased, or full agonists—can alter the composition of the cognate protein network and thus change the overall cellular response to GPCR activation. Here we sought to capture these cellular dynamics at the level of local GPCR protein interaction networks and elucidate differences between ligands. To do so, we employed an approach which combines: (1) sub-minute proximity labeling to capture proteins near a GPCR in living cells, (2) mass spectrometry to determine protein identity and quantify abundance across time and different subcellular locations. We have focused on the mu-type opioid receptor (MOR) and three opioid agonists— DAMGO, morphine, and PZM21—to examine what protein network components are shared across all ligands, and what networks components are agonist-specific? Consistent with previous studies, we observed proximity-labeling of the G protein with all three agonists while only the full agonist DAMGO caused strong labeling of B-arrestin. We corroborated this observation by using our proximity-labeling approach to track the relative location of MOR within cells after agonist stimulation and found that only the full agonist drove efficient endocytosis of the receptor. We then examined the networks for previously unidentified proteins which might function with MOR. We identified two proteins—labeled by MOR with all three agonists tested—which fit the profile of novel regulators of MOR signaling through the heterotrimeric G protein. Future studies will examine differences in networks and test the newly identified network components for a role in mediating and regulating opioid receptor function.
The central melanocortin system is critical to maintenance of energy homeostasis. The melanocortin-4 receptor (MC4R), a component of this system, is a G-protein coupled receptor that regulates both energy intake and expenditure. Therefore, MC4R is a target for drug development in the treatment of obesity. The MC4R couples to Gas, thought to be the classical pathway through which the receptor signals. However, our lab has recently shown that there are alternative signaling pathways that may play an important role in the normal function of the receptor, including allosteric modulation of MC4R activity by the melanocortin-2 receptor accessory protein 2 (MRAP2), which regulates the receptor’s constitutive activity. Many GPCRs are also known to recruit beta-arrestin upon activation which leads to downstream intracellular events, however, there have been few systematic studies to date examining beta-arrestin recruitment by the MC4R. Mutations in the MC4R are the most common cause of syndromic obesity, and multiple obesity-associated mutations in the MC4R gene either disrupt trafficking of the receptor to the plasma membrane or coupling to Gas. However, up to a third of obesity-associated MC4R mutations produce receptors that appear normal by these tests. We hypothesize that these receptor mutations may specifically alter functions of MC4R signaling not detected through the study of Gas coupling. We created expression vectors for a collection of 16 obesity-associated mutant MC4Rs reported to have normal Gas coupling. These receptors were then characterized using assays for 1) Gas coupling, 2) binding to and suppression of constitutive activity by MRAP2, 3) receptor glycosylation, and 4) beta-arrestin recruitment. One mutant exhibited aberrant Gas coupling, and was not studied further. Most remaining mutants exhibited abnormal beta-arrestin recruitment in comparison with WT receptor, while maintaining normal Gas coupling. One mutant (V50M) exhibited both abnormal glycosylation and beta-arrestin recruitment. These data argue that there are signaling properties beyond Gas coupling, such as beta arrestin recruitment, that are crucial for normal physiological function of the MC4R.
Phospholipase Cβ (PLCβ) enzymes hydrolyze the lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to produce the second messengers inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). Production of these second messengers leads to many diverse physiological responses, including vascular smooth muscle contraction and inflammation. Previous structural and functional studies of PLCβ have revealed a highly conserved catalytic core that adopts a compact, globular structure and forms the minimal fragment that retains lipase activity. However, a growing body of evidence suggests that the PLCβ PH domain, which interacts with EF hands and TIM barrel of the catalytic core in crystal structures, may be flexible in solution. Using a combination of small angle x-ray scattering (SAXS), cross-linking studies, and biochemical assays, we provide the first structural data demonstrating that the PH is in fact conformationally flexible in solution. The PH domain adopts two major states: an open state wherein the PH domain is extended away from the core, and a closed state consistent with the crystal structures. We also provide evidence that these conformational states are functionally significant. These findings provide new evidence of conformational heterogeneity of PLC enzymes in solution and reveal new insights into their roles in cell signaling and cardiovascular disease.
The C-terminal guanine exchange factor module of Trio (TrioC) serves as a link between the heterotrimeric G proteins Gαq/11 and the small G-protein RhoA, joining Gαq/11-coupled GPCRs to downstream events of cell motility and gene transcription. This ancient signal transduction axis has been identified as crucial to the rise and spread of the fatal malignancy uveal melanoma. Previous studies have shown that TrioC is likely regulated via an autoinhibitory constraint that is released upon binding of Gαq/11. However, the structural determinants of this autoinhibition remain unclear. We have determined the crystal structure of TrioC in its basal state, revealing high-resolution detail of autoinhibition mediated by the Pleckstrin Homology (PH) domain. We show that truncation of the PH domain activates the module in vitro, showcasing the importance of the αN helix found at the DH/PH junction. We then show using two orthogonal biochemical assays that the disruption of the autoinhibited conformation by point mutations destabilizes and activates the module. Finally, we show that mutations in αN found in cancer patients hyperactivate TrioC. These point mutations increase mitogenic signaling through the activation of RhoA in cells and may thus represent the first known cancer drivers identified in Trio, operating in an analogous, yet Gαq/11 independent manner. We are in the process of confirming this hypothesis using cell-based assays.
Uveal melanomas are the most prevalent eye tumor, arising from melanocytes of the uveal tract. These tumors are characterized by activating mutations in GNAQ and GNA11, 2 subunits of Gαq/11 heterotrimeric G proteins, and in PLCB4. In analyzing genomic data from 136 uveal melanomas, we identified a new c.386T>A mutation in CYSLTR2 gene encoding in a Leu129Gln substitution1. This mutation is mutually exclusive with the GNAQ/11 or PLCB4 mutations in uveal melanoma.
CYSLTR2 encodes a G protein-coupled receptor (GPCR), cysteinyl leukotriene receptor 2 (CysLT2R). Leu129 is located in the transmembrane helix 3 of cysLT2R, and resides in a highly conserved 3.43 position. To characterize L129Q cysLT2R signaling, we measured its ability to couple to Gαq (in measuring calcium flux mobilization and Inositol phosphate (IP1) accumulation, a product upstream of calcium flux), Gαs and Gαi2 (in measuring ligand-mediated increase in cAMP levels (Gαs) or decrease of forskolin-induced cAMP levels (Gαi2)) in HEK293T cells expressing the WT or L129Q cysLT2R. The WT cysLT2R mobilizes calcium flux1 and IP1 accumulation in a dose-dependent manner in response to leukotriene D4 (LTD4) agonist, while L129Q cysLT2R revealed a high basal calcium flux mobilization1 and IP1 accumulation, and is mainly unresponsive to LTD4. We didn’t show any cAMP increase or decrease activity with neither the WT or Leu129Gln cysLT2R, nor any alteration of the basal level. These results indicate that L129Q cysLT2R is a Gαq constitutively active and is not coupled to Gαs and Gαi.
Otherwise, expression of L129Q cysLT2R in murine melanocytes enforces the expression of melanocyte-lineage-specific genes (like Mitf or Kit), drives phorbol ester (TPA)-independent growth in vitro (while TPA is usually required for melanosome growth and pigmentation) and enhances melanoma tumorigenesis in vivo1. These data together demonstrate supportive evidence of this mutation being a new oncogenic driver.
The use of a Gαq/11 inhibitor, YM254890, allowed us to totally inhibit the downstream IP1 constitutive accumulation promoted by L129Q cysLT2R, in a dose-dependent manner, in both HEK293T cells and melan-a cells. Besides, none of the known antagonists of cysLT2R or cysLT1R were able to show any significant inhibitory effect on L129Q cysLT2R constitutive activity.
In summary, our findings identified a first GPCR, CYSLTR2, as a driver oncogene in uveal melanoma. Therefore, L129Q cysLT2R is a potential therapeutic target for uveal melanoma therapy.
1Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in uveal melanoma. Nat Genet. 2016 Jun;48(6):675-80. Moore AR, Ceraudo E, Sher JJ, Guan Y, Shoushtari AN, Chang MT, Zhang JQ, Walczak EG, Kazmi MA, Taylor BS, Huber T, Chi P, Sakmar TP, Chen Y.
The many pharmacological functions of the opioid receptors make them interesting subjects of study and targets of interest for many indications. The role of the opioid system in regulating the rewarding properties of drugs of abuse demonstrates potential for opioids to be used in the treatment of addiction. It has been previously demonstrated that kappa opioid receptor (KOR) agonists have the potential to reduce cocaine self-administration in non-human primates. However, KOR activation is also associated with negative side effects such as dysphoria which limit the clinical utility of these ligands. It has been suggested that mu opioid receptor (MOR) partial agonism may be able to mitigate dysphoria associated with KOR agonism, increasing the therapeutic potential of a KOR agonist. We have developed a series of novel KOR agonist/MOR partial agonist ligands based on the classically delta opioid receptor (DOR) antagonist selective dimethyltyrosine-tetrahydroisoquinoline (DMT-Tiq) scaffold. Installation of a 7-benzyl pendant on the tetrahydroisoquinoline aromatic ring introduced strong KOR agonism. A series of further modifications led to the development of several structurally similar KOR agonist/MOR partial agonist compounds with slightly different pharmacological profiles. These ligands are currently being evaluated for their in vivo effects in mice.
Proteinase-activated receptor 4 (PAR4) is a member of the proteolytically activated PAR family of GPCRs. PAR activation occurs through proteolytic cleavage of the receptor N-terminus by enzymes such as thrombin, trypsin, and Cathepsin-G to unmask a receptor activating tethered ligand sequence. In addition to proteases, PARs can be activated by tethered-ligand mimicking synthetic peptides. For the PAR4 receptor, the tethered ligand sequence revealed by thrombin cleavage is GYPGQV while the most potent tethered ligand derived agonist peptide is AYPGKF-NH2. To determine the structure-activity relationship (SAR) for PAR4 activation by the tethered ligand peptide we generated a library of AYPGKF-NH2 derivatives and identified key determinants of PAR4 dependent Gαq/11 coupling and β-arrestin activation. We further investigated the PAR4 SAR through in silico peptide docking studies to identify key receptor-peptide interactions (GalaxyPepDock). in silico docking studies revealed several receptor residues predicted to engage AYPGKF-NH2. To study this further, we mutated the most highly predicted peptide-receptor interacting residues- D230, H229, and Q242- and examined Gαq/11-coupling (Fluo4 calcium signaling) and β-arrestin recruitment (BRET) to activated PAR4 in response to AYPGKF-NH2 and thrombin. Interestingly, predicted sites of interaction between PAR4 and AYPGKF-NH2 impacted activation of G-protein signaling and β-arrestin recruitment with both AYPGKF-NH2 and thrombin. We find that PAR4D230 is an essential residue for Gαq/11-coupling and β-arrestin recruitment with both AYPGKF-NH2 and the native tethered ligand. Interestingly, mutation of PAR4Q242 had no effect Gαq/11-coupling or β-arrestin recruitment with AYPGKF-NH2 but did decrease signaling in response to the tethered ligand mediated receptor activation. Through these studies, we have elucidated key residues in the PAR4 orthosteric ligand binding pocket to support further studies aimed at identifying novel PAR4 agonists and antagonists.
The adhesion GPCRs (AGPCRs) GPR56 and GPR114 belong to a receptor sub-class distinguished by the presence of an extracellular GPCR autoproteolysis inducing (GAIN) domain that is sufficient to self-cleave the receptor into two fragments. AGPCR fragment dissociation reveals a hidden peptide that serves as a tethered-peptide-agonist that presumably binds intracellularly to the GPCR fragment to initiate G protein signaling. To understand how agonist peptides interact with their cognate receptors, we developed a photo-crosslinking approach to map the interaction sites between P7 (TYFAVLM) and P18 (TYFAVLMQLSPALVPAEL) agonist peptides and GPR56 and GPR114. Analogue agonist peptides were synthesized that contained the unnatural crosslinking amino acids BzP (4-Benzoyl-L-Phenylalanine) or AzP (4-Azido-L-Phenylalanine) at positions required for agonist activity. These will be used in UV-light-dependent reactions to crosslink to orthosteric binding site residues that lie with 3-4 Å distance. To optimize conditions for crosslinking and detection of the peptide-receptor complexes, we used synthetic agonist peptides conjugated to biotin [P7(AzP or BzP)-biotin and P18(AzP or BzP)-biotin]. With these biotinylated probes we demonstrated UV-dependent probe crosslinking to membranes prepared from cells that expressed the receptor. Immunoblot analyses showed UV-dependent bands when probed with fluorescently-tagged streptavidin conjugates at the molecular mass of the receptor. This demonstrates formation of covalent binding (crosslinking) between P7 (AzP or BzP)-biotin and GPR56, and between P18(AzP or BzP)-biotin and GPR114. A larger library of synthetic peptide crosslinking probes was tested to identify specific probes that retained receptor agonistic activity using SRE- and CRE- luciferase cell-based assays. We identified one crosslinkable peptide probe for GPR56 and GPR114 that had comparable agonistic activity to the unmodified peptide agonist. Here, we have developed a methodology to probe the binding interface between AGPCRs and synthetic peptide agonists. The application of our findings will lead to the identification of AGPCR orthosteric binding sites and aid the pharmacological design of small molecule AGPCR inhibitors and activators.
The human Y4 receptor is a G-protein coupled receptor (GPCR) that is expressed predominantly in gastrointestinal tract. Y4 and its native ligand, pancreatic polypeptide (PP), play critical roles in regulating feeding behaviors and energy expenditure. Hence, selective allosteric modulators (AM) of Y4 can serve as promising therapeutics for obesity and eating disorders. Because the allosteric binding sites tend to be less conserved than the orthosteric counterparts, small-molecule allosteric modulators have been in focus of research to discover novel GPCR ligands with high subtype selectivity. Therefore, the objective of this study is to discover novel AMs for Y4 through virtual screening, and then develop those confirmed hit compounds into small molecule in vitro probes of the Y4 receptor. We combine ligand-based (LB) and structure-based (SB) computer aided drug design (CADD) approaches to expedite probe development. LB virtual screening such as quantitative structure-activity relationship (QSAR) can quickly narrow libraries of millions of drug-like molecules to hundreds of compounds candidates for experimental validation. Our QSAR models, which were trained on an input set of known 19 active Y4 positive allosteric modulators (PAMs) and 80,000 inactive compounds, were used to rank predicted PAM activity of 150,000 compounds in Vanderbilt internal database. Out of top 600 compounds with highest predicted activity, 20 compounds showed significant PAM property in Ca2+ flux-based bioassays. Moreover, two novel Y4 PAM scaffolds were discovered. On the other hand, SB methods, one of which is ligand docking, characterize the interaction between AM hits and Y4 homology models. To obtain insight into the antagonistic mechanism, VU0637120—a Y4 selective antagonist, was docked to Y4 homology models. Computational docking of VU0637120 identified an allosteric binding site located below the binding pocket of the endogenous ligand pancreatic polypeptide (PP) in the core of the Y4R transmembrane domain. This study will improve the general understanding of allosteric modulation, ultimately contributing to the rational development of allosteric modulators for peptide-activated GPCRs.
The human dopamine receptors D2R and D3R are both representatives of the largest group of G-protein coupled receptors (GPCRs), namely the rhodopsin-like receptors. The future goal of our project is the investigation of ligand-receptor interactions and dynamics of GPCRs by nuclear magnetic resonance (NMR) spectroscopy. To study D2R and D3R by NMR spectroscopy we are establishing heterologous expression systems as well as stabilization and purification protocols to obtain mg-amounts of pure and active isotope labelled protein necessary for biophysical experiments. We were able to successfully express both receptors in E. coli, insect cells as well as in an E. coli based cell-free expression system. Currently, we are optimizing the expression and stability of the receptors by a directed evolution based approach. Therefore, we are generating a library of receptor variants using random mutagenesis by error-prone PCR. The library will be expressed in E. coli. After labelling of the receptors with a fluorescent ligand, highly fluorescent cells can then be sorted by a fluorescence activated cell sorter (FACS). This allows screening for more stable and highly expressed receptor variants. Furthermore, GPCR variants being well expressed and stable in E. coli have been shown to exhibit similar behavior in other heterologous expression systems. A purification protocol will be established based on previous work on other GPCRs which involves the incorporation of the receptors into nanodiscs. Subsequently, we will analyze ligand binding to D2R and D3R by NMR spectroscopy. In collaboration with the group of Prof. Gmeiner (University of Erlangen-Nürnberg) the interaction of new D2R/D3R selective agonists, antagonists, inhibitors, etc. with the receptors will be analyzed. NMR experiments will help to understand conformational changes of the dopamine receptors upon ligand binding and will reveal conformational differences of the transmembrane helices upon ligand binding.
The CXC motif chemokine receptor 4 (CXCR4), and its cognate chemokine CXCL12, play essential roles in hematopoiesis, cardiogenesis and immune responses. In addition, CXCL12 and CXCR4 are linked to several diseases, including cancer. CXCR4 signaling contributes to several aspects of tumor progression yet it remains poorly understood. A comprehensive understanding of CXCR4 signaling is of great therapeutic significance. The present study is focused on a novel β-arrestin-dependent signaling pathway regulating chemotaxis by CXCR4. Previous work from our lab showed that β-arrestin1 (βarr1) interacts directly with signal-transducing adaptor molecule 1 (STAM1) to promote CXCR4-dependent chemotaxis. Expression of fragments from either βarr1 or STAM1 disrupts the βarr1/STAM1 interaction and significantly attenuates CXCL12-dependent chemotaxis and activation of focal adhesion kinase (FAK). Together, these data support the hypothesis that the βarr1/STAM1 complex activates FAK to promote chemotaxis by CXCR4. However, mechanistic insight is lacking. Here, we address the hypothesis that STAM1 induces a unique conformational signature in βarr1. To examine this we employed double electron-electron resonance (DEER) EPR (Electron Paramagnetic Resonance) spectroscopy. By incorporating spin labels on selected double cysteine mutants of βarr1, DEER can detect intramolecular conformational changes in βarr1 by measuring distances between spin labeled residues. We monitored the inter-spin distance changes on 3 selected pairs of double βarr1 cysteine mutants upon binding to activated and phosphorylated GPCR (pGPCR*) or STAM1. We observed predicted conformational changes on βarr1 induced by pGPCR* binding, including finger loop extension, C-tail displacement and middle loop shift. STAM1 binding induced a similar finger loop extension on βarr1, however, in contrast to pGPCR*, there was no C-tail displacement or middle loop shift. These data indicate that βarr1 undergoes unique conformational changes upon STAM1 binding, likely because STAM1 binds to βarr1 at a site that is different from the pGPCR* site. Next, we mapped the STAM1 binding site on βarr1 by CW-EPR (Continuous Wave) spectroscopy. By monitoring the CW-EPR spectral line-shape changes on select spin labeled single cysteine βarr1 mutants in the presence and absence of STAM1, it allowed us to assess whether the spin labeled residue is involved in STAM1 binding. We identified two spin labelled residues located on the back side of βarr1 that showed significantly immobilized motion upon STAM1 binding, indicating their contribution to the interaction with STAM1. Importantly, pGPCR* or other non-GPCR βarr1 binding partners do not bind to these sites. In conclusion, STAM1 binding induces unique conformational changes in βarr1, which we propose is coupled to discrete functional outcomes, such as promoting FAK activation and ultimately chemotaxis by CXCR4.
The delta-opioid receptor (DOR) has become a promising alternative target for pain management. However, DOR localizes intracellularly in sensory neurons resulting in the requirement of high doses of DOR agonists for pain relief. Thus, delivery of these retained intracellular receptors to the plasma membrane increases the efficacy of DOR agonists. Nevertheless, the retention mechanism of these intracellular pool of DORs remains poorly understood. We have identified two ‘RXR’ motifs within the C-terminus tail of DOR that are required for retention in the trans-Golgi network (TGN). Additionally, we find that these motifs bind to Beta-COP, a component of COPI retrograde trafficking machinery, in vivo and in vitro. We hypothesize that the overexpression of the C-terminal tail can overwhelm the retention machinery and consequently decrease the intracellular pool of DORs. We found that overexpression of a TGN anchored DOR tails shows a significant decrease in DOR retention in the Golgi. Further experiments will determine if this effect is specifically caused by the RXR motif.
The prostaglandin F (FP) receptor plays and important role in parturition and is a potential target for delaying preterm birth. In myometrial cells, its natural ligand, PGF2α, is a strong inducer of labor via the canonical Gaq/11 and Ga12/13 signaling pathways. Therefore, dissecting the physiological output of each signaling pathway at the FP receptor could potentially help generate better tocolytics (e.g. labor repressants). Here, we used a high-throughput mutagenesis strategy to complete an alanine scan of the 359 amino acids in the FP receptor, and further screened in HEK293 cells their signaling behaviors for Gaq/11 and Ga12/13 activation using bioluminescence resonance energy transfer (BRET) biosensors. We identified ~20 residues in FP, which did not impact receptor expression, but significantly affected Gaq/11 signaling ability, while also maintaining WT levels of Ga12/13 signaling. Conversely, 9 residues in FP were found to impede Ga12/13 signaling ability without affecting Gaq/11 signaling. Combinatorial mutation in FP allowed us to generate both Gaq/11–null, Ga12/13 signaling and Ga12/13 –null, Gaq/11-signaling receptors. Our finding reveal specific residues important for the preferential G-protein coupling of FP receptor, which might explain selectivity of GPCR signaling. The functional selectivity of the combinatorial FP receptor mutants should help in dissecting out the relevant physiological contribution of Gaq/11 and Ga12/13 signaling pathways at the FP receptor.
Opioid addiction and accidental overdose have arisen as critical health issues as the prevalence and availability of this addicting class of drugs have dramatically increased. The mu opioid receptor (MOR) is the primary analgesic mediator in the brain and is activated by the class of drugs known as opioids. MOR is a G protein coupled receptor (GPCR) that signals through the heterotrimeric G protein class Gi/o. Once activated, the heterotrimeric G proteins transduce the opioid signal to other effectors including adenylate cyclase, voltage-gated calcium channels, and G protein-coupled inward-rectifying potassium (GIRK) channels, among others. Together, this signaling renders the cell less excitable and blocks transmission of pain responses but also elicits unwanted side effects. Endogenously, MOR signaling is regulated to terminate or tune signaling and hence to alter the duration or intensity of the opioid response. Regulators of G protein signaling (RGS) proteins, for example, serve as GTPase-activating proteins to inactivate Gi/o and terminate MOR signaling. Receptors such as the neuropeptide FF (NPFF) receptor and the cholesystokinin type B (CCKB) receptor, are reported to exhibit “anti-opioid” effects. We hypothesized that there may be undiscovered negative MOR regulators that could modulate MOR signaling to lessen opioid side effects. We are currently developing an unbiased forward genetic screen in c. elegans to uncover potential new cell-autonomous MOR modulators. While the screen is an ongoing effort, we are working to establish the framework for evaluating potential hits in cell-based assays of MOR signaling. As proof of concept, we examined the effect of the known negative MOR regulator R7BP-RGS7-Gb5 complex in cellular assays of MOR function. Overall, this project is aimed at uncovering and determining the molecular mechanisms of potential new anti-opioid signaling pathways that could be pharmacologically modulated to mitigate negative opioid-related side effects.
GPCRs coupled to Gαq, stimulate phospholipase Cβ (PLCβ), inducing hydrolysis of phosphatidylinositol 4, 5 bisphosphate (PIP2) by activating Gq heterotrimers. Resultant products; diacylglycerol (DAG) and inositol triphosphate (IP3) regulate crucial cellular functions by activating protein kinase C (PKC) and mobilizing stored calcium. Gq (GNAQ) pathway involves in crucial functions including transcription regulation, immune responses, cell growth, and learning and memory. GNAQ also identified as a cancer driver gene by genome wide cancer screening. It has been shown that Gq pathway-induced elevated cytosolic calcium could be harmful and cells employ several adaptation mechanisms such as receptor desensitization and calcium sequestration & efflux to attenuate signaling. Here we show that, most cell types employ a tight expression control of GNAQ/11 to achieve transient signaling upon Gq pathway activation. We further demonstrate that cells transfected with GNAQ or with higher levels of endogenous GNAQ/11 exhibit a distinct signaling regime characterized by adaptation-resistant PIP2 hydrolysis. Elevated Gq expressions are associated with enhanced Gβγ signaling, an uncommon signaling modality in Gq pathway. We also show that GNAQ is overexpressed in matched normal samples of patients having Kidney Chromophobe (KICH) and Kidney renal papillary (KIRP) cell carcinomas. Collectively, these data indicate a Gαq expression regulation-dependent distinct signaling regimes which is not reported for Gαi/o as well as Gαs mediated pathways.
G protein-coupled receptors (GPCRs) mediate neuronal responses to neurotransmitters and neuromodulators, and are major drug targets in neuropsychiatric disease. Individual GPCRs may signal via multiple downstream effectors, only some of which may mediate therapeutic effects in vivo. Furthermore, the specific complement of signalling cascades engaged by a given GPCR is determined by several factors, including the particular ligand, as well as the cellular and tissue context. In order to study GPCR mediated signalling in its native context in vivo we have developed tools for expressing and recording Förster resonance energy transfer (FRET)-based biosensors that report signalling downstream of GPCRs in real time in live animals. Our approach uses adeno-associated virus (AAV) to express genetically encoded sensors in wild-type animals. By using cell-type selective promoters and Cre-recombinase dependent promoters, we can target biosensor expression to desired neuronal populations in vivo. Biosensor responses are recorded from live animals using a fiber-photometry-based FRET recording platform. Using a FRET based reporter of protein kinase-A activity we show real time recording of PKA activation in the dorsal striatum in response to D1 dopamine receptor stimulation.
Chemokines, chemokines analogs, and small molecules can prevent cellular HIV-1 entry when bound to the HIV-1 coreceptors CCR5 or CXCR4. CCR5 is the primary target for HIV-1 transmission, and high potency chemokine analogs that block HIV-1 entry using CCR5 are being test in clinical trials as topical preventative agents. In developing topical anti-HIV agents, it is crucial to prevent CCR5-dependent immune response, and therefore, the ideal HIV-1 entry blocker targeting the coreceptors would display ligand bias and avoid activating G protein-mediated pathways that lead to inflammation. We studied the signaling of two endogenous chemokines, RANTES and MIP-1α, and four chemokine analogs, 5P12-, 5P14-, 6P4- and PSC-RANTES, and compared different CCR5-dependent second messenger pathways. We could demonstrate that all six ligands are agonists for Gi-dependent inhibition of forskolin-induced cAMP formation, and that the effect is pertussis toxin (PTX)-sensitive. Using PTX and the Gq-specific inhibitor YM-254890, we discovered that the CCR5 signaling-dependent Ca2+flux and IP1 accumulation arose from Gq/11 activation, rather than from Gβγ subunit release after Gi/o activation as had been previously proposed. PSC- and 6P4-RANTES are superagonists relative to RANTES and MIP-1α on the Gq-pathway, whereas 5P12- and 5P14-RANTES are antagonists. We demonstrate that the chemokine analogs elicit G protein subtype-specific signaling bias and can cause CCR5 to couple preferentially to Gq/11 rather than to Gi/o signaling pathways. We propose that GPCRs coupling to two or more G protein families can display G protein-specific bias and that agonist ligands can be designed to bias GPCRs to signal through specific G protein signaling pathways.
 E. Lorenzen, E. Ceraudo, Y.A. Berchiche, C.A. Rico, A. Fürstenberg, T.P. Sakmar, T. Huber. G protein subtype-specific signaling bias in a series of CCR5 chemokine analogs. Science Signaling, in press. (2018)
The Gαs subunit is classically involved in the signal transduction of G-protein-coupled receptors (GPCR) at the plasma membrane. However, recent evidence has revealed the presence of Gαs on endosomes and its non-canonical role in endosomal sorting of receptors to lysosomes. Yet, the mechanism of action of Gαs in this sorting step is still poorly characterized. Because ubiquitin is key in the endosomal sorting pathway, we investigated whether Gαs interacts with ubiquitin.
In cellulo and in vitro GST-pull-down assays were used to investigate the interaction between Gαs and ubiquitin. The specific residues implicated in this interaction have been mapped by mutagenesis and NMR titrations. Confocal microscopy and western blot analysis were used to investigate sorting into intraluminal vesicles of endosomes for lysosomal degradation.
Gαs interacts directly with ubiquitin and colocalizes with ubiquitinated proteins in microdomains of early endosomes. A functional and conserved ubiquitin-interacting motif (UIM) was identified at the N-terminal extremity of Gαs. Most importantly, mutation of the UIM in Gαs prevented the transfer of EGFR (a classical example of ubiquitinated receptors) into intraluminal vesicles, leading to an accumulation of EGFR on the limiting membrane of endosomes, and thus, the delay of its degradation.
Although Gαs structure has been known for years, we found a novel motif in Gαs that allows its interaction with ubiquitin, a key signal for cargo sorting to the lysosomal pathway.These findings demonstrate a new role for Gαs as an integral component of the ubiquitin-dependent endosomal sorting machinery and highlight the dual role of Gαs in receptor trafficking and signaling for the fine-tuning of the cellular response.
GPR56 (ADGRG1) is a member of a subset of G protein coupled receptors called adhesion GPCRs (AGPCRs). AGPCRs possess an extracellular GPCR autoproteolysis-incuding (GAIN). This domain catalyzes an autoproteolysis event, splitting the receptor into two, noncovalently bound fragments whose dissociation can lead to receptor activation. GPR56 couples to G12/13 family G-proteins and its dysfunction causes the neurodevelopmental disorder bilateral frontoparietal polymicrogyria (BFPP). Pilot high-throughput screens were conducted to find agonists and antagonists of GPR56. HEK293T cells were transfected with either a truncated receptor with compromised activity (GPR56 A386M) or a fully-active receptor (GPR56 7TM) to screen for agonists and antagonists, respectively. The serum response element luciferase (SRE-Luc) gene reporter was co-transfected to provide readout of G-protein signaling. Hits from the screen were vetted in counterscreens utilizing either constitutively active Gα13-Q226L or reporter-only transfections. This screen identified a first-in-class GPR56 partial agonist, 3-α-acetoxydihydrodeoxygedunin (3αDOG) and an antagonist, dihydromunduletone (DHM). Our goal is to expand to a large-scale screen at the Center for Chemical Genomics (CCG). Large-scale HEK293T transfections (1 x 109 cells) were optimized for signal-to-noise ratios by calculating Z’ assay quality scores. Cells with optimal Z’-scores will be screened in conjunction with the CCG. Hits that survive directed counterscreen testing will be verified by directed dual luciferase assays and an in vitro GPCR reconstitution assay. The discovery of high affinity agonists or antagonists will be useful probes to explore AGPCR activities in tissue and organ systems. Additionally, these probes will be vital for solving AGPCR structures – none of which currently exist. Eventually, these molecules may even serve as leads for development of AGPCR-directed therapeutic compounds.
In vitro biochemical assays have been invaluable in characterizing signaling and spectral properties of retinal binding G protein coupled receptors (opsins). Since subcellular environment imposes additional regulatory mechanisms and distinct structural-organizational support or constraints for signaling activation and regulation by opsins, their signaling interrogation in living cells is mandatory. Here we examined the optogenetic potential of two mammalian and one-invertebrate opsins, two of which are bistable. Results show that human SW opsin is an efficient activator of Gi/o pathways and utilizes existing 11-cis retinal dissolved in the lipid bilayer or 11-cis retinal formed by blue light-induced isomerization of free all-trans retinal, to form the inactive and yet blue light activatable opsin. Since this cone opsin is monostable, it is unable to use ultra-low retinal concentrations in cell culture media and recycle it to generate a detectable level of G protein activation. On the contrary, the invertebrate lamprey parapinopsin induced a detectable level of G protein heterotrimer activation indicating its bistability and the ability of chromophore recycling. Our data also show that while parapinopsin interacts with all-trans retinal, contrary to the current understanding, in the dark this all-trans retinal-bound opsin exhibited no signaling activation. Our data also show that, parapinopsin is a better trigger for subcellular optogenetics applications due to its low retinal requirement and large spectral window that allows multiple biosensor imaging without activating the opsin.
The mu opioid receptor (MOR) is the primary target for opioids prescribed to treat pain. We sought to understand how opioid-induced signaling may regulate MOR trafficking. Specifically, we focused on MOR recycling back to the cell surface, which affects recovery of sensitivity and acute tolerance. We hypothesized that signaling downstream of MOR can modulate its own recycling. To test this, we used total internal reflection fluorescence microscopy (TIR-FM) to visualize and quantify individual MOR exocytic recycling events in HEK293 cells, as a measure of recycling without the confounding effects of other trafficking events such as endocytosis. Continued activation of MOR by the presence of agonist increased the rate of MOR recycling, suggesting that MOR recycling is regulated by a feedback loop. G protein signaling via Gβγ was required and sufficient for this feedback, via activation of a Phospholipase-C/Protein Kinase C pathway that phosphorylated serine 363 on the C-terminal tail of MOR. This represents a novel route of receptor regulation that could serve as a model for how signaling could regulate post-endocytic trafficking of GPCRs.
G-protein coupled receptors (GPCRs) are an important class of cell surface receptors that transduce extracellular stimuli to intracellular signaling events via G-proteins. One major effector of G proteins is phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). PLCβ2 and PLCβ3 isoforms are directly activated by binding Gαq, Gβγ or both. The mechanism of activation of PLCβ2 by Gβγ is unknown, and the binding site(s) for Gβγ on PLCβ remain controversial.
Here we use hydrogen-deuterium exchange mass spectrometry (HDXMS) to understand the conformational dynamics of PLCβ and to answer open questions in the field concerning the mechanism of activation of PLC by Gβγ and Gαq. For these experiments, several complexes were examined with HDXMS: PLCβ2 alone, PLCβ2+ PIP2/phosphoethanolamine (PE)/phosphatidylserine (PS) vesicles, or PLCβ2+ PIP2/PE/PS vesicles plus Gβγ or Gαq-AlF4. Incubation of PLCβ2 with PIP2/PE/PS vesicles strongly protects regions in the C-terminus, confirming that this region is involved in membrane binding. In the presence of Gαq-AlF4, PLCβ2 shows protection from deuterium exchange in the helix-loop-helix region of the proximal C-terminal domain (CTD) and the EF-hand domain on PLCβ2 that exactly correspond to regions shown to interact with Gαq in the Gαq-PLCβ3 crystal structure. These data validate that our approach accurately reports bona fide protein-protein and protein-lipid interactions. Incubation of Gβγ with PLCβ2 causes strong increases in deuterium exchange in the CTD of PLCβ2, indicating that the CTD of PLC undergoes a large conformational rearrangement upon Gβγ binding. This increase in deuterium exchange in the CTD of PLC is not seen upon Gαq activation. Additionally, in a purified, in vitro system, CTD-deleted PLCβ2 displays increased responsiveness to Gβγ and isolated CTD can inhibit PLCβ2 activity. Taken together, these data suggest a mechanism for activation of PLCβ by Gβγ, where Gβγ binding to PLCβ breaks an autoinhibitory interaction involving the CTD of PLC and either the membrane or another portion of PLCβ2 and that Gαq and Gβγ use different mechanisms for activation of PLCβ.
G protein betagamma is a major signal transducer and controls processes ranging from cell migration to gene transcription. Despite the significant subtype heterogeneity and diverse cell-tissue specific expressions, Gbetagamma is often considered a unitary signaling entity. Bearing the only plasma membrane (PM) anchoring motif, we find Ggamma subunits control the PM-affinity of Gbetagamma, thereby differentially modulate Gbetagamma effector signaling in a Ggamma-type dependent manner. We also demonstrate that macrophage migration requires high PM-affinity Ggamma for sufficiently intense signaling activation. Results also indicate that the overall PM - affinity of Ggamma types in a cell is a strong predictor of the efficacy of Gbetagamma signaling. The kinetic model tested encapsulated multiple Ggamma types and a signaling loop containing translocating Gbetagamma between the PM and internal membranes, reflecting the natural Gbetagamma distribution and behavior. Overall, our data unveil a crucial regulation of G protein signaling and cell behavior by Ggamma-type specific PM affinities of Gbetagamma subunits.
Angiotensin receptor 1 (AT1R) is key effector for the renin-angiotensin system that regulates blood pressure and fluid balance. Renal AT1R modulates a multitude of kidney functions and is expressed in renal vasculature, glomeruli and proximal tubules and is believed to be primarily responsible for Ang II-mediated hypertension. G protein-coupled receptor kinase (GRK) dictates GPCR desensitization and uncouples the activated GPCRs from their downstream signaling effectors. G protein-coupled receptor kinase 2 (GRK2) was the only isoform of the seven known GRKs that caused embryonic lethality after homozygous deletion in mice suggesting that it is the most crucial isoform. GRK2 modulates AT1R desensitization and its expression was found to be altered in hypertensive patients. We employed shGRK2 knockdown mice (shGRK2 mice) to test the role of GRK2 in kidney development and function that can be ultimately linked to the hypertensive phenotype detected in shGRK2 mice. shGRK2 mice present with reduced kidney size and impaired electrolyte balance. GRK2 knockdown altered nephrogenesis and was associated with reduced glomerular count and impaired glomerular filtration rate in adult mice. We also detected glomerular sclerosis in adult shGRK2 mice that was associated with increased renin- and AT1R-mediated production of reactive oxygen species. No compensatory change from other isoforms of GRK (GRK3,4,5,6) was observed indicating that these isoforms do not contribute to developmental/functional changes in shGRK2 mice. The AT1R blocker, Losartan, normalized elevated blood pressure and markedly improved glomerular filtration in the shGRK2 knockdown mice. Together, our data provide evidence for the crucial role of GRK2 in renal mechanisms of blood pressure homeostasis and indicate that total inhibition of GRK2 can have detrimental outcomes on kidney function and development if not carefully monitored.
Hypertension and its associated risks can be attributed to the endogenous hormone angiotensin II (AngII), which regulates blood volume and vascular resistance through the AngII type 1 receptor (AT1R),a member of the G protein-coupled receptor (GPCR) superfamily.As a GPCR, the AT1R signals by coupling to G proteins (Gαq/11, Gα12/13, Gαi1/2/3) and β-arrestins (β-arrestin1 and 2, which act both as endocytic and signaling adaptors) to mediate its physiological effects.Mutations in the AGTR1 gene (encoding the AT1R) that alter the native amino acid sequence are referred to as nonsynonymous mutations (NSMs). While certain NSMs of the AT1R have been reported to reduce receptor expression and AngII binding, it is unknown whether mutations can also alter downstream cellular responses. Therefore in this study, we investigated the effects of three NSMs of the AT1R (A163T, T282M, and C289W) on signaling and trafficking. We hypothesized that the amino acid alterations caused by these NSMs will induce unique receptor conformations that would allow for differential coupling to cognate G proteins and/or β-arrestins. We used an array of bioluminescence resonance energy transfer (BRET)-based biosensors to quantify the receptors’ efficiency for engaging β-arrestins to undergo internalization and trafficking relative to the wild type AT1R. Results show that upon AngII stimulation, the A163T displayed increased β-arrestin-mediated receptor endocytosis relative to the WT, while the T282M and C289W had reduced relative activities in all the pathways assessed. Notably, although the T282M recruited β-arrestin and internalized upon AngII stimulation, the receptor trafficked into early endosomes without being in complex with β-arrestin. Consequently, the recycling rate of the T282M from the endosomes back to the plasma membrane is drastically increased. Next, when we probed the conformational profile of β-arrestin binding to the receptors using β-arrestin2-FlAsH BRET sensors, we found significant differences between the T282M and the WT AT1R in the conformational signatures of β-arrestin binding. When we looked at β-arrestin-mediated signaling, we found that T282M was completely deficient in β-arrestin-dependent MAPK activation as compared to the WT-AT1R. Finally, we used HEK293 cells and β-arrestin-knockout HEK293 cell lines to test the WT and T282M on AT1R-dependent cellular responses, such as proliferation and migration. We found that for the T282M, which display weak avidity for β-arrestin and a deficiency in β-arrestin-dependent signaling, AngII does not promote AT1R-dependent proliferation or migration responses for the T282M. On the other hand, while AngII induced both cell proliferation and migration in the WT AT1R, these responses were not observed in the β-arrestin-knockout cells transfected with the WT. Taken together, these results suggest the importance of β-arrestin-mediated signaling in AT1R-dependent cell migration and proliferation, and suggest that the T282M mutation confers a conformation in the AT1R that alters the conformation and avidity of β-arrestin binding, highlighting the importance of the T282 position of the AT1R for regulating β-arrestin-mediated trafficking and signaling.
A systems view of G protein–coupled receptor (GPCR) signaling in its native environment is central to the development of GPCR therapeutics with fewer side effects. Using the kappa opioid receptor (KOR) as a model, we employed high-throughput phosphoproteomics to investigate signaling induced by structurally diverse agonists in five mouse brain regions. Quantification of 50,000 different phosphosites provided a systems view of KOR in vivo signaling, revealing novel mechanisms of drug action. Thus, we discovered enrichment of the mechanistic target of rapamycin (mTOR) pathway by U-50,488H, an agonist causing aversion, which is a typical KOR-mediated side effect. Consequently, mTOR inhibition during KOR activation abolished aversion while preserving beneficial antinociceptive and anticonvulsant effects. Our results establish high-throughput phosphoproteomics as a general strategy to investigate GPCR in vivo signaling, enabling prediction and modulation of behavioral outcomes.
G protein-coupled receptor kinases 2 and 5 (GRK2 and GRK5) are expressed in the heart and have been implicated in playing a detrimental role during heart failure. Previously, we discovered two classes of GRK2-selective inhibitors, one stemming from GSK180736A and the other from paroxetine, and have synthesized over 100 derivatives based on these scaffolds. From this library, seven inhibitors were identified that demonstrated high potency (IC50 < 1mM) and selectivity for both GRK2 and GRK5 over related kinases. Compounds with a dual potency for GRK2 and GRK5 could prove advantageous because they would prevent one kinase from compensating for the other and could inhibit multiple maladaptive processes that are reliant on specific GRK isoforms. Five of these dual-potency inhibitors have been crystallized in complex with GRK2-Gβγ, which revealed, through principal component analysis, a kinase-domain conformation similar to that observed in a GRK5-inhibitor complex. The ability of GRK5 to adopt an analogous kinase-domain conformation could at least partially explain the loss of GRK2 selectivity and gain in GRK5 potency observed for these particular inhibitors. In order to determine if inhibition of both GRK2 and GRK5 would be advantageous physiologically, we are using a live-cell receptor internalization assay to compare a panel of GRK2-selective inhibitors to GRK2/5 dual-potent inhibitors. Internalization of the μ-opioid receptor and β2-adrenergic receptor were assessed in HEK293 and U2OS cells, which display differential expression of GRKs. Several of the dual-potency GRK2/5 inhibitors tested have higher efficacy in preventing internalization of the μ-opioid receptor in U2OS cells, which express GRK5 in much higher levels than HEK293 cells. Therefore, these two cell lines have enabled us to assess the relative importance of GRK5 in promoting internalization of GPCRs.
Metabotropic glutamate receptors (mGluRs) are a family of class C G-protein coupled receptors found highly expressed throughout the central and peripheral nervous system. These receptors modulate neuro-excitability through various g-protein signaling cascades in response to the neurotransmitter glutamate. The family is composed of 8 members (mGluR1-8) each of which form homodimers to create a functional receptor complex. Recently, increasingly convincing data has surfaced to support the idea that mGluRs can also heterodimerize with a specific pattern. There is however, no proposed molecular mechanism for how these proteins determine which heterodimers complexes can or cannot form. Here we explore this selectivity using whole cell voltage clamp, microscopy, and sequence and structural analysis in an effort to explain the molecular determinants and cellular control over the surface expression of functional mGluR heterodimers.
Latrophilin-3 (ADGRL3) is an adhesion G protein coupled receptor (AGPCR) enriched in synaptic membranes. Latrophilins (1-3) are important for development of synapses and maintenance of synaptic integrity. The mechanism(s) by which Latrophilins become activated and the downstream G protein pathways that they may activate are unclear. Latrophilins are typical AGPCRs that are self-cleaved at the conserved GPCR-autoproteolysis-inducing (GAIN) domain site ~20 residues N-terminal to the start of the first transmembrane span (TM1). We hypothesize that one mechanism of Latrophilin activation is via a tethered-peptide-agonist unmasking process that requires dissociation of its N-terminal and C-terminal fragments. To test our hypothesis, we reconstituted Latrophilin-3-enriched membranes with representative members of all four heterotrimeric G protein subfamilies (Gq, G13, Gi, Gs) and measured the kinetics of receptor-stimulated G protein GTPgS binding. Latrophilin-3 is shown to robustly couple to G13, exhibited appreciable Gi and Gq coupling, but did not activate Gs. Dramatic enhancement of G protein activation was observed when the Latrophilin-3 NTF was dissociated, indicating that the release of the tethered-peptide-agonist from the GAIN domain hydrophobic core was critical for receptor activation. We then serially truncated single, N-terminal residues of the Latrophilin-3 CTF tethered-peptide-agonist and tested the engineered receptors for activation of the G13-dependent Serum Response Element (SRE)-luciferase gene reporter. The first three N-terminal Latrophlin-3 CTF residues were critical for signaling. A small peptide library comprising the Latrophilin-3 tethered-peptide-agonist was synthesized and tested for the ability to activate Latrophilin-3. Latrophilin-3 synthetic peptides comprising sequences N’-TNFAVLM(Xn), but not GPR56/114 derived peptides comprising sequences N’-TYFAVLM(Xn) activated Latrophilin-3. Our work demonstrates that Latrophilin-3 is activated via a tethered-peptide-agonist mechanism, which may be a general paradigm for AGPCR activation. Ongoing work is examining our new finding that Latrophilin-3 is predominantly coupled to G13. This may provide important new insight into the role Latrophilins have in shaping synaptic architecture.
The central melanocortin receptors, including the melanocortin 3 receptor and the melanocortin 4 receptor, are G-protein coupled receptors that regulate energy homeostasis. The critical role of the MC4R in controlling energy homeostasis is well established. However, until recently the precise role of central MC3R in energy homeostasis and related behaviors remained elusive. Recent work from our lab has outlined a specific role for the melanocortin-3 receptor in the process of energy homeostasis (MC3R; Ghamari-Langroudi et al., Science Advances 2018). In particular, we demonstrated that the MC3R is required for control of the upper and lower boundary conditions in the process of homeostatic set point regulation. These data implied a potential role for the MC3R in energy rheostasis, or the regulated movement of set point. We also reported in this paper that the MC3R was required for normal energy acquisition during pregnancy, a challenge that involves a regulated increase in food intake and energy stores. During fasting, the hypothalamic-pituitary-gonadal (HPG) axis is suppressed, and this is mediated in part by activation of hypothalamic AgRP neurons. Here we show that the MC3R is required for normal activation of hypothalamic neurons and suppression of the HPG axis by fasting. Normal mice exhibit a robust activation of c-fos in arcuate nucleus neurons following a fast, while MC3R knockout mice do not. Furthermore, while normal mice will terminate reproductive cycling for up to a week following a 16hr fast, mice with deletion of the MC3R exhibit normal cycling under these same conditions. Collectively, these data show that MC3R is critical for coordinating bidirectional interactions between reproductive and metabolic state.
Parkinson’s Disease (PD) is a progressive neurodegenerative disease characterized by the loss of dopaminergic neurons in the nigral striatal pathway and presence of Lewy body aggregates. These pathological hallmarks lead to a variety of motor and non-motor symptoms and there are currently no disease-modifying or neuroprotective therapeutic options. Evidence suggests regulation of metabotropic glutamate receptor 5 (mGluR5) signaling can positively affect pathological outcomes in preclinical neurodegenerative disease models such as Alzheimer’s Disease (AD), Huntington’s Disease (HD), and PD. While there are many inducible preclinical models for PD, it is challenging to best model the disease as the underlying mechanism(s) for neurodegeneration in PD is unknown. Recent literature has focused on eliciting Lewy body like pathology by promoting the production and spread of alpha-synuclein (α-Syn). Here, we developed an inducible mouse model to elicit widespread α-Syn pathology by pairing an α-Syn WT adeno virus (AV) with a preformed firbril (PFF) injection, four weeks apart. This model was used to explore the effectiveness of CTEP, an orally available negative allosteric modulator (NAM) for mGluR5 on behavioural and pathological features. C57BL/6 mice aged 10-12 weeks were subject to either an EGFP injection followed by a saline injection or an α-Syn WT AV injection followed by a PFF injection into the substantia nigra pars compacta (SNc). CTEP dosing (2 mg/Kg) began one week prior to the second injection and continued every 48 hours for 18 weeks. Chronic treatment with CTEP significantly protected motor performance on rotarod, limb coordination on the horizontal ladder, and locomotor activity in open field tests and cognitive dysfunction in object recognition task. Chronic CTEP treatment also protected against α-Syn accumulation and loss of dopaminergic neurons in the striatum (TH+ fibre density). These findings provide evidence for future use of this novel mouse model can effectively recapitulate PD pathology. It also further evidence that mGluR5 NAM should be considered as potential therapeutic approach for PD patients.
Alzheimer’s disease (AD) is characterized by neurotoxicity mediated by the accumulation of beta amyloid (Aβ) oligomers, causing neuronal loss and progressive cognitive decline. We have previously showed that genetic deletion or chronic pharmacological inhibition of mGluR5 by the negative allosteric modulator CTEP, rescued cognitive function and reduced Aβ aggregation in male APPswe/PS1ΔE9 (APPswe) mouse model of AD. We have recently described the novel ZBTB16-medaited autophagic mechanism downstream of mGluR5 that plays a key role in Huntington’s disease (HD) progression in zQ715 mice. We also showed that the favorable outcomes of mGluR5 silencing in male APPswe were associated with a reduction in ZBTB16 similar to zQ175 mice. Here, our recent results indicate that, unlike male mice, chronic (12 week) treatment with CTEP of female APPswe mice does not improve AD pathology. We tested brain samples from male and female APPswe mice following 12-week treatment with either vehicle or CTEP (2mg/kg) for changes in the autophagic clearance of Aβ oligomers via ZBTB16 pathway. When quantified using ELISA, both male and female APPswe mice exhibited higher level of Aβ oligomers at 6 months of age. To our surprise, CTEP significantly reduced Aβ burden in male mice yet, it exacerbated Aβ oligomers accumulation in female APPswe mice. mGluR5 blockade in male APPswe mice caused a GSK3β-dependent degradation of ZBTB16 and stabilization of the autophagy adaptor ATG14. This was paralleled by a reduction in the autophagy marker p62. Interestingly, when we tested the same pathway in female mice we did not detect any change in GSK3β phosphorylation, ZBTB16, ATG14 or p62 levels between APPswe and control mice. It is worth noting that female APPswe brain also exhibited lower expression of mGluR5 compared to age-matched male mice. This study provides evidence that mGluR5 inhibits autophagy via GSK3β/ZBTB16 pathway that can result in impaired clearance of Aβ burden in male APPswe only. We also provide clear evidence, and for the first time, that aspects of mGluR5 signaling are not conserved between males and females and there are sex-specific differences that must be considered when embracing mGluR5 as a potential drug target for neurodegenerative disease.
Previously, our laboratory identified a phospholipase C (PLC)-mediated PI4P hydrolysis pathway at the Golgi apparatus that is important for regulation of cardiac hypertrophy, a pathology associated with development of heart failure. cAMP stimulates this pathway via Epac-mediated activation of Rap1 which directly binds to PLCe. We recently found that a cell membrane permeable b-adrenergic receptor (b-AR) agonist, dobutamine, induces Golgi PI4P hydrolysis in neonatal rat ventricular myocytes (NRVMs) while a cell membrane impermeable b-adrenergic (b-AR) agonist, Isoproterenol, does not (see accompanying poster by Nash et al.). This suggests that an internal pool of b-ARs may mediate activation of this EPAC-PLCe pathway at the Golgi. To directly determine if internal b1-ARs in NRVMs can be activated by exogenously applied ligands, we used live cell fluorescence microscopy to monitor translocation of a YFP-tagged mini Gs protein in b1-AR transfected NRVMs. The mini-Gs protein developed by Wan et al 2 is recruited to Gs-coupled receptors upon their activation, providing readout of the location of activation of these receptors. Dobutamine induced robust and rapid recruitment of mini Gs to both the plasma membrane, and the perinuclear region corresponding to the Golgi apparatus. Isoproterenol, on the other hand, induced a strong recruitment to the plasma membrane but not to the Golgi. Treatment with brefeldin A significantly reversed mini Gs recruitment to the internal membrane induced by dobutamine, supporting the idea that active b1-ARs are localized to the Golgi membrane. A membrane permeable b-AR antagonist, metoprolol, fully reversed dobutamine mediated mini Gs recruitment while a membrane impermeable antagonist, sotalol only partially reversed the mini Gs recruitment to the Golgi. The physiological b-AR agonist norepinephrine, also induced a robust recruitment of mini Gs to the plasma membrane and the Golgi. These observations indicate the presence of a Golgi localized pool of b1-ARs in cardiac cells similar to that previously demonstrated in HeLa cells 3. These data support other data from our laboratory indicating that activation of endogenous intracellular b1ARs stimulates production of a local pool of cAMP that activates the perinuclear Epac-PLCe-PI4P hydrolysis pathway involved in heart failure.
The cellular context in which GPCRs are found is a key driver of their biological activities. Countless biosensor based in-cell assays aim to better characterize signaling downstream of GPCRs as with a view toward designing better drugs. With improvements in fluorescent probe chemistry, conjugation of receptors with fluorescent reporter proteins allows for the generation of specific tools that capture particular endpoints. Signaling biosensors monitoring specific biological readouts (Ca2+, cAMP, phosphorylation etc.) have proven their utility for drug discovery. However, differences in the genetic backgrounds of diverse cell types might bias the resulting signaling phenotypes as a direct consequence of cell context. Here, we propose to make use of conformation-sensitive biosensors that we have developed as tools for drug research and discovery in more relevant cellular contexts. To build a biosensor line that reports on conformational changes within GPCRs, we introduced fluorescent biarsenical hairpin binder sites (FlAsH) as acceptors for bioluminescence resonance energy transfer (BRET). We inserted multiple FlAsH tags along the intracellular surface of the AT1R and β2AR along with a C-terminally fused Renilla luciferase to monitor the conformation of the receptors upon ligand binding. By exploring receptor conformation, the effect of cell context is restricted to interacting proteins present at the membrane bypassing unwanted noise generated as a consequence of signal amplification. To explore cell context with disease relevance, we will use inducible pluripotent stem cells as a model due to their capacity to differentiate into any cell lineage. Our conformation-sensitive biosensors will be introduced into the AAVS1 safe harbour site of an engineered iPS cell line and subsequently differentiated into cardiomyocytes, vascular smooth muscle cells as well as neurons. To date, we have successfully generated beating cardiomyocytes that express the cardiomyocyte markers troponin T and α-actinin. Once validated, our system using receptor conformation as a readout can guide rational drug discovery as we hypothesize that our sensors will be able to discriminate different types of agonists by potency and efficacy in disease relevant cellular backgrounds.
Despite the wealth of genetic information available,
mechanisms underlying pathological effects of disease-
associated mutations in components of G protein-
coupled receptor (GPCR) signaling cascades
remain elusive. In this study, we developed a scalable
approach for the functional analysis of clinical
variants in GPCR pathways along with a complete
analytical framework. We applied the strategy to
evaluate an extensive set of dystonia-causing mutations
in G protein Gaolf. Our quantitative analysis
revealed diverse mechanisms by which pathogenic
variants disrupt GPCR signaling, leading to a mechanism-
based classification of dystonia. In light of
significant clinical heterogeneity, the mechanistic
analysis of individual disease-associated variants
permits tailoring personalized intervention strategies,
which makes it superior to the current phenotype-
based approach. We propose that the platform
developed in this study can be universally applied
to evaluate disease mechanisms for conditions
associated with genetic variation in all components
of GPCR signaling.
Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids to produce diacylglycerol (DAG) and inositol phosphates, to activate protein kinase C (PKC) and downstream signaling pathways, including cell growth and survival. The PLCepsilon (PLCε) subfamily has emerged as a key player in cardiovascular health, where it is required for maximum contractility. However, prolonged activation of PLCε results in cardiac hypertrophy and heart failure through its ability to regulate hypertrophic gene expression. This process is regulated by the small GTPase Rap1A, which is activated downstream of β-adrenergic receptors. Rap1A binds to the C-terminal Ras association (RA) domain of PLCε, simultaneously translocating the complex to the perinuclear region and activating PLCε. However, the molecular mechanism of this process is not known. We seek to characterize the interactions between Rap1A and PLCε using structural and functional studies to map the Rap1A binding site on PLCε and determine whether activation results in conformational changes that release autoinhibition and/or increase membrane association. These studies provide the first molecular details of the Rap1A-dependent activation of PLCε and opens the door to the development of new therapeutic strategies for treating cardiac hypertrophy.
The Melanocortin Receptor Accessory protein 2 (MRAP2) regulates the activity of multiple G-Protein Coupled Receptor (GPCR) involved in the regulation of food intake and energy homeostasis. The Growth Hormone Secretagogue Receptor-1a (GHSR1a or ghrelin receptor) is a GPCR that couples to Gαq. It is an important regulator of food intake and glucose homeostasis. We have previously shown that GHSR1a signaling is potentiated by the Melanocortin Receptor Accessory Protein 2 (MRAP2) and that MRAP2 is required for the function of ghrelin on orexigenic AgRP/NPY neuron. In this study we investigated the effect of MRAP2 on the pharmacology of GHSR1a. We tested the effect of MRAP2 on ghrelin binding, ghrelin-stimulated β-arrestin recruitment and GHSR1a signaling. Using radiolabeled ligand-receptor binding assay we show that MRAP2 does not alter GHSR1a affinity for ghrelin. Using a nanobit luminescence recombination assay we measured ghrelin stimulated β-arrestin recruitment to GHSR1a and found that MRAP2 significantly inhibits this process. This finding was confirmed by microscopy. This study shows that MRAP2 potentiates GHSR1a signaling through Gαq and inhibits ghrelin-stimulated β-arrestin recruitment, thus suggesting a potential role of MRAP2 in biasing GHSR1a activity.
Morphine and other opiates remain among the most effective treatment option of both severe and chronic pain. However, the utility of these compounds in the clinical setting is limited because of the rapid development of tolerance, physical dependence and addiction. While the underlying cellular and molecular alterations through which chronic opiate exposure results in the persistent behavioral phenomenon of addiction remain poorly understood, it is known that the effects of opiate drugs are mediated by the μ-opioid receptor (MOR). After binding to opiate drugs the MOR activates intracellular signaling through inhibitory G proteins leading to the dissociation of heterotrimeric G proteins into Gα and Gβγ subunits, which in turn regulate downstream ‘‘effector’’ molecules. Here, we present two novel mediators of MOR signaling in the striatum which produce opposing effects on reward-related behaviors; regulator of G protein signaling 7 (Rgs7) and neurofibromin 1 (NF1). Rgs7 acts as GTPase activating proteins (GAP) to stimulate GTP hydrolysis on the Gα thereby catalyzing their inactivation. Lack of striatal RGS7 increased the sensitivity of rewarding and reinforcing behaviors to morphine without affecting analgesia, tolerance, and withdrawal. We also show that NF1, a major Ras regulator is a direct effector of MOR signaling via Gβγ subunits in the striatum. In contrast to Rgs7, striatal ablation of NF1 sensitizes mice to the rewarding effects of morphine. With cellular resolution we were able to determine the cell-specific manner in which NF1 contributes to these reward-related behaviors. Overall, we seek to demonstrate the impact of intracellular signaling pathways commonly targeted by MOR and their distinct contributions to morphine-induced behaviors.
G-protein coupled receptors (GPCRs) are a diverse set of transmembrane proteins with significant structural variations across classes. These variations are likely to affect spatial organization and may help explain why GPCR dimerization/oligomerization is still not fully understood. Here, we focus on two classes of GPCR: class A (rhodopsin-like) and class C (glutamate-like) with the goal of measuring oligomerization for representative members. Specifically, we present on applying new controls to previously reported data on cone opsin constructs. The class C metabotropic glutamate receptor (mGluR) is known to exist as a dimer due to presence of disulfide bond formation in the extracellular cysteine-rich domain (CRD), serving as positive dimer control. Removal the extracellular domain of mGluR then serves as a monomer control. Each control is stably transfected in a Chinese Hamster Ovary (CHO) cell line and expressed via the tetracycline inducible TRex system. A SNAP-tag, which allows for covalent attachment of SNAP-tag dyes, is fused to the N-terminus of each GPCR control. Expressed proteins are labeled using both green (SNAP Surface 488) and red (SNAP Surface 549) fluorescent dyes. Our lab utilizes a custom-built pulsed interleaved excitation-fluorescence cross correlation spectroscopy (PIE-FCCS) setup. With this method, 488 nm and 561 nm lasers are focused onto the peripheral membrane of live cells, exciting green and red tags respectively. Fluorescence fluctuations are then recorded and analyzed to determine the extent to which green and red tagged proteins co-diffuse through the confocal volume indicating the degree of interaction between species. The single-molecule sensitivity provided by PIE-FCCS allows us to determine dimerization affinity in a quantitative manner.
Introduction: β-arrestins are multifunctional proteins involved in regulation of G protein-coupled receptors (GPCR). They are mainly associated with the termination of G protein–dependent signalling pathways, or with the scaffolding of different complexes, such as the endocytic machinery and G protein independent signalling. These roles may be interdependent: for example, desensitization is often followed by receptor internalization. It has also been proposed that some of β-arrestin-dependent but G protein-independent signalling may not require internalization. However, the lack of selective pharmacological tools that would allow discrimination between the different roles of β-arrestins hinders the study of their specific actions.
In order to study the spatiotemporal roles of β-arrestins, Dr. Bouvier's team identified in 2017 a small chemical molecule that selectively inhibits the interaction between β-arrestins and AP2, a clathrin-adaptor protein essential for the initiation of β-arrestin–dependent endocytosis through clathrin-coated pits (CCP). The inhibitor binds on the binding site of β-arrestin on the β2-adaptin subunit of AP2, thus selectively competing with this interaction. Yet, this molecule, called Barbadin, has relatively low potency and efficacy, as well as poor solubility, which limits its use.
Objectives: This present project has for objective to increase the solubility, the efficacy and the potency of Barbadin, by optimizing its chemical structure.
Methodology: Based on the docking mode of Barbadin in AP2, 20 analogs were designed, synthesized, and tested for their ability to inhibit AP2–β-arrestin1 interaction. Only one analog (#23) showed higher efficacy and potency. Functional characterization of this analog was essential to confirm that the properties that make Barbadin a good pharmacological tool were conserved. Thus, its ability to inhibit the interaction between AP2 and the two isoforms of β-arrestin, and consequently to inhibit β-arrestin–dependent internalization through CCP, without altering their recruitment to activated receptors, was evaluated using BRET-based assays.
Results: The analog 23 showed better efficacy and potency than Barbadin to inhibit the interaction between AP2 and both β-arrestins, with also a better potency to inhibit internalization of vasopressin receptor-2 (V2R). Direct and indirect methods of detection for the recruitment of β-arrestin to activated V2R showed contradictory results.
Conclusion: We succeed to synthesize a new analog of Barbadin that had better potency and efficacy to inhibit β-arrestins–AP2 interaction, which lead to more potent inhibition of internalization. However, our results concerning the effect of Barbadin on β-arrestin recruitment to activated receptors are ambiguous. More explorations are required in order to elucidate this paradox.
Optineurin (OPTN) is a cytosolic protein that is involved in multiple cellular functions such as; membrane trafficking, inflammatory response and autophagy. OPTN interactome involves a variety of binding partners including metabotropic glutamate receptors (mGluR5), a member of Gq/11 protein-coupled receptor family. We have previously reported that mGluR5, in addition to its canonical G protein-mediated signaling, triggers cellular pathways that help cell maintain balance between cell survival and autophagy. In attempt to investigate the role of OPTN in mGluR5-mediated regulation of autophagy and cell survival, we knocked out Optn gene in human embryonic kidney cells (HEK-293) using CRISPR/Cas9 approach. Successful OPTN knockout in HEK293 cells was confirmed by western blotting and genomic sequencing and two OPTN clones were selected for our experimental design (C1 and C2). Both basal and quisqualate-stimulated IP3 formation were reduced in mGluR5-transfected OPTN knockout cells compared to mGluR5-transfected control cells. The loss of OPTN disrupted autophagic flux in cells as demonstrated by high levels of P62. Moreover, OPTN knockout cells exhibited lower expression of phosphoinositide 3-kinase (PI3K) compared to control cells, an effect that was coupled with marked inhibition of its downstream targets, Akt and 4E-BP1. Quisqualate partially rescued the activation of 4E-BP1 in mGluR5-transfected compared to non-transfected OPTN knockout cells. Taken together, the data suggest that OPTN is crucial for mGluR5 canonical Gq/11-signaling and IP3 formation. Moreover, the results provide evidence for the novel role OPTN as an adaptor protein for the regulation of PI3K/Akt/mTOR pathway via mGluR5.
Opioid analgesics are the gold standard for treating chronic and severe pain. However, they are plagued with negative side effects, including tolerance, dependence, abuse liabilities, constipation, and death through respiratory depression. There is a growing body of evidence that suggests agonism at the µ-opioid receptor (MOR) with concomitant antagonism at the δ-opioid receptor (DOR) can induce opioid mediated analgesia with reduced or abolished negative side effects. To this end, our lab has developed a peptidomimetic series with the above profile that induce antinociception in vivo without tolerance, dependence, or drug-seeking behavior. Despite these favorable attributes, these ligands express poor stability in mouse liver microsome assays. As such, we have opted to pursue an SAR campaign aimed at improving this stability. This SAR campaign has produced compounds that express our desired MOR-agonist/DOR-antagonist profile, are stable in mouse liver microsomes with half-lives of over an hour, and show antinociception in vivo. Herein, the SAR campaign that produced these more stable ligands will be described.
Heart disease is one of the leading causes of death in the United States. Cardiac fibrosis is commonly associated with heart diseases, such as cardiac hypertrophy and myocardial infarctions. This is characterized as an excess deposition of extracellular matrix that results in a stiffening of the heart. Cardiac fibrosis can lead to arrhythmias, decreased diastolic function and eventually heart failure. Upon injury to the heart, cardiac fibroblasts transition to an activated myofibroblast state, characterized by the expression of α-smooth muscle actin (αSMA). While the activation of fibroblasts is initially beneficial to the heart, chronic activation of fibroblasts leads to excessive deposition of collagen resulting in the development of fibrosis. Phospholipase Cε (PLCε) is an important signaling enzyme that is activated by small GTPases downstream of GPCRs and receptor tyrosine kinases and is important in the cardiac myocyte for the development of cardiac hypertrophy. In this study we sought to elucidate the role of PLCε in cardiac fibroblasts. Using neonatal rat cardiac fibroblasts, we demonstrate that PLCε is highly expressed in fibroblasts compared to myocytes as assessed by Western blot. Its expression is equally high in fibroblasts and myofibroblasts. Adenovirus encoding for a PLCε-shRNA was used to knockdown PLCε in fibroblasts and the ability of the cells to transition to a myofibroblast was examined. Knockdown of PLCε decreases the ability of cardiac fibroblasts to transition to myofibroblasts assessed by staining for αSMA in untreated cells or cells treated with TGFβ. In addition, knocking down PLCε decreases the mRNA expression of profibrotic genes such as αSMA and TGFβ. This indicates that PLCε may have an important role in regulating the myofibroblast transition and that PLCε may also be involved in TGFβ signaling.
Traditional opioids produce analgesia by acting at the orthosteric site on the µ-opioid receptor (MOR); however, GPCR function can also be controlled by compounds acting at a separate (allosteric) site on the receptor. One compound, BMS-986122, binds to the allosteric site on the MOR and enhances the affinity and potency of MOR agonists in vitro. We hypothesized that BMS-986122 will produce antinociception by enhancing endogenous opioid activity in vivo. Mice were administered drugs via intraperitoneal injections (1-10 mg/kg; i.p.). Antinociception was assessed using the warm water tail withdrawal test and the von Frey test after induction of hindpaw inflammation using 2.5% carrageenan (20 µL) or Complete Freund’s Adjuvant (5 µL). Constipation was assessed by counting the number of fecal boli deposited in two hours. Tolerance was assessed by measuring antinociception after repeated administration of drug. BMS-986122 (10 mg/kg) produced near-maximal antinociception on the tail withdrawal test for 2 hours, which was reversed by a 20 min pretreatment with naloxone (10 mg/kg). BMS-986122 reversed carrageenan-induced mechanical allodynia 30 min post-injection, which was also reversed by naloxone pretreatment. The antinociceptive effect of BMS-986122 against carrageenan-induced allodynia persisted after 3 injections of drug. BMS-986122 produced antinociception 24 hours post-CFA and continued to produce antinociception after 5 injections of drug. Lastly, mice treated with morphine deposited fewer fecal boli compared to mice treated with BMS-986122. The findings suggest that positive allosteric modulation of the µ-opioid receptor produces antinociception that is naloxone-reversible in models of acute and inflammatory pain suggesting it is likely mediated via enhancement of endogenous opioid activity in vivo. BMS-986122 maintains efficacy against inflammatory pain with repeated administration and does not constipate mice compared to morphine. Positive allosteric modulation of the µ-opioid receptor provides a safe and effective approach for treating pain by enhancing the body’s endogenous opioid activity without side effects.
Metabotropic glutamate receptors (mGluRs) are class C G protein coupled receptors with widespread expression in the central nervous system. Unlike many GPCRs, mGluRs exist and function as obligate, disulfide linked dimers. While all mGluRs can form homodimers, and many can heterodimerize, we have recently published a paper demonstrating that while mGluR1 and 5 may reside in close proximity to each other, and can interact functionally, this interaction does not appear to be due to canonical heterodimerization. Pharmacological evidence supporting this conclusion is largely pharmacological. For example, in the presence of both receptors, the selective mGluR1 competitive antagonist 3-MATIDA produced little inhibition where strong block would be expected in heterodimers. The selective negative allosteric modulators for mGluR1 and mGluR5, BAY36 7620 and MPEP, respectively, strongly inhibit the signal when both receptors are expressed, where only weak inhibition would be expected with heterodimers. To ask whether the observed mGluR1/5 interaction was due to a physical, allosteric interaction between the receptors rather than integration of downstream signaling effects, point mutations in each receptor were introduced that uncouple the receptors from G protein signaling, presumably without affecting ligand binding or associated conformational changes. We found that when mGluR1 was co-expressed with a double point mutant of mGluR5, Y64A F768D (which right-shift the glutamate response and uncouple from G proteins, respectively), the glutamate dose-response of mGluR1 was right shifted. Further, when mGluR1Y74A, a mutant with a 300 fold right-shifted glutamate response, was expressed with mGluR5 F768D, the response was significantly left shifted. Expression of the signaling dead mGluR5 F768D with mGluR1 also altered the pharmacological sensitivity profile, causing mGluR1 to lose sensitivity to 3-MATIDA and gain sensitivity to MPEP. Together, these findings support the hypothesis that allosteric interactions between the receptors are responsible for the observed functional interactions.
The prostaglandin F2α receptor (FP) is expressed in the myometrium and is an important regulator of myometrial contraction. Thus, it serves as a target to prevent preterm labor. Recent studies have shown the propensity of FP to be allosterically modulated to bias its signaling. Interestingly, selective inhibition of the Rho/Rock signaling cascade downstream Gα12/13 while potentiating ERK1/2 and PKC downstream of Gαq/11 showed a promising therapeutic advantage in vivo, by delaying spontaneous and induced contractions in mice. This bias in FP receptor signaling could be driven by a competitive binding at the level of G proteins; where the potentiation of Gαq/11 leads to an inhibitory effect on Gα12/13 activity. To address this, we looked at the level of G protein coupling using a panel of Bioluminescence resonance energy transfer (BRET)-based biosensors in CRISPR-Cas9 Gαq/11 and Gα12/13 Knockout HEK293 cells. We show that Gαq coupling on FP and the activation of its downstream mediators; PKC and MAPK were unaffected by the deletion of Gα12/13. On the other hand, coupling of FP to Gα12 and Gα13 was significantly potentiated in the absence of Gαq/11 activity. More interestingly, Gα13 coupling was inhibited upon the addition of Gαq in a dose dependant manner. This effect was further assessed for potential feedback mechanism through a Gαq/11 intermediate downstream effector; PKC and we show that PKC had no involvement in modulating the ability of FP to couple Gα12/13. These results imply that Gα12/13 coupling could be suppressed directly by the receptor’s ability to couple to Gαq/11. Further establishment of the mode of competitive regulation between G proteins at FP would help explain the mechanisms driving signaling bias and ultimately aid in the design of better therapeutics.
The location of GPCRs dictates their differential signaling responses. Post-endocytic sorting is key to regulating location and controlling spatial encoding of signaling. Although spatial encoding provides an attractive model for new strategies for drug development, how this process is regulated is not well understood. The β2-adrenergic receptor (β2AR) is a physiologically relevant model to study this. After agonist-mediated internalization, β2AR is restricted to a spatially distinct signaling domain on the endosome. This restriction required multiple C-terminal sequences - a retention sequence preventing it from bulk recycling and a recycling sequence blocking it from degradation. To identify the machinery that restricts β2AR, we performed stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry to determine the candidate proteins that binds to β2AR’s retention sequence. The present study focused on the role of endofin, a signaling scaffold protein, in β2AR endosomal sorting and signaling. We used endofin siRNA and shRNA in cells expressing β2AR, and analyzed endosome number, β2AR containing tubules, rate of internalization, recycling, and expression of PCK1, a marker of β2AR signaling from endosomes. Our results suggest that endofin is a key factor ensuring proper localization of β2AR to specialized endosomal signaling domains. Further understanding of this regulation may define principles that are generalizable to regulation of spatial encoding of signaling by other GPCRs.
Ghrelin is an essential hormone for survival during starvation periods. It is secreted by the stomach during low energy state to promote food intake and maintain normal glycemia. The mechanisms through which ghrelin increases blood glucose involve the stimulation of growth hormone release from the pituitary and the inhibition of insulin secretion by pancreatic β-cells. The way that ghrelin suppresses insulin release is however poorly understood. It was recently shown that, within the endocrine pancreas, the ghrelin receptor (GHSR1a) is exclusively expressed in the somatostatin secreting δ-cells. Based on this finding, ghrelin is thought to stimulate somatostatin release from δ-cells which in turn inhibits insulin release from β-cells. We have previously shown that GHSR1a signaling is enhanced by the Melanocortin Receptor Accessory Protein 2 (MRAP2). In this study we show that MRAP2 is, like GHSR1a, expressed in pancreatic δ-cells. We also found that the deletion of MRAP2 results in a decrease in ghrelin-stimulated somatostatin secretion from δ-cells as well as a loss of ghrelin-mediated inhibition of insulin secretion. These results demonstrate that MRAP2 is essential for the normal function of GHSR1a in the pancreatic islet and identifies MRAP2 as a novel modulator of insulin secretion.
Resistance to Inhibitor of Cholinesterase-8 (Ric-8) proteins play essential roles in regulating heterotrimeric G protein a subunits. Ric-8A and Ric-8B are molecular chaperones that assist the folding of nascent Ga subunits by facilitating first-time binding of GTP to Ga. Ric-8 proteins also catalyze Ga subunits guanine nucleotide exchange (GEF) in vitro. The Ric-8A in vitro GEF activity is possibly a manifestation of the chaperoning function of Ric-8 to fold Ga subunits in a conformation that can properly bind GTP. We recently identified multiple protein kinase CK2 consensus phosphosites in Ric-8A. Three of the phosphosites, Ser-522, Ser-523 and Ser-527 are located at the Ric-8A C-terminus, which have no known function. A structural prediction of Ric-8A indicated that five phosphosites, including Ser-522, Ser-523, Ser-527, and two previously characterized residues Ser-435 and Thr-440, reside closely in three-dimensional space in a distinct C-terminal ~100 amino acid domain. The central Ga binding domain is the core, N-terminal ~430 residue armadillo repeat region. We hypothesize that the C-terminal domain functions as a flexible latch to facilitate high affinity Ga binding. To investigate the potential function of Ser-522, Ser-523 and Ser-527, we produced stoichiometrically phosphorylated wildtype and mutant Ric-8 proteins at > 99% purity using an in vitro protein kinase CK2 phosphorylation procedure. Ric-8A mutant proteins with alanine substitution or truncation of Ser-522, Ser-523 and Ser-527 are around 40% slower than WT Ric-8A in stimulating Gaq GTPgS binding. However, mutations of Ric-8A Ser-522, Ser-523 and Ser-527 phosphosites did not affect binding affinity to Gaq, suggesting that the decreased GEF activities of Ric-8A mutants were not due to hampered Ric-8A-Gaq association. The Ric-8A C-terminal CK2 consensus sequence is also present in Ric-8B. Mass spectrometry analysis of Ric-8B identified Ser-556 phosphorylation, which is equivalent to Ric-8A Ser-523. We conclude that the phosphorylation of Ric-8 conserved serine/threonine residues in the C-terminal domain enhances Ric-8-stimulated Gaq GTPgS binding. Phosphorylation of Ric-8A C-terminal residues is required for the C-terminal latch to efficiently fold Gaq to a conformation that allows GTP binding.
Phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchanger (P-Rex) is a Rho guanine-nucleotide exchange factor that regulates cell motility through its activation of small GTPases such as Rac1 and Cdc42. P-Rex is synergistically recruited to the cell membrane and activated by PIP3 and Gβγ subunits, positioning the enzyme downstream of multiple classes of cell surface receptors including GPCRs and receptor tyrosine kinases (RTKs). P-Rex1 functions as a critical regulator of neutrophil migration, but aberrant upregulation of P-Rex1 is strongly associated with cancer metastasis, and as such it has become an attractive therapeutic target. Development of selective inhibitors against P-Rex1 is hindered by the fact that its overall architecture and regulatory mechanisms are poorly understood. Thus, we have taken a multi-pronged structural biology approach using X-ray crystallography, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and cryo-electron microscopy (EM) to define the molecular basis for the regulation of P-Rex1, with the aim of identifying its important regulatory surfaces and mechanisms of activation. We determined crystal structures of the P-Rex1 tandem Dbl homology (DH)/pleckstrin homology (PH) domain catalytic core and showed that the PH domain is necessary and sufficient for PIP3-dependent activation. Our data are consistent with PIP3 activating P-Rex1 via an allosteric mechanism. Using these structures, along with data from HDX-MS and cell-based experiments, we developed a model for P-Rex regulation wherein another domain within P-Rex1 binds to the DH and PH domains, resulting in a condensed tertiary structure that occludes the substrate-binding site on the DH domain. To test this model, we are analyzing cryo-EM data of P-Rex1 alone and in complex with regulatory molecules with the goal of providing high-resolution information for the full-length, 183 kDa protein. To this end, we have generated cryo-EM data on a P-Rex1–Gβγ complex to ~4 Å resolution. Our data support a condensed state for P-Rex1 that is consistent with our HDX-MS experiments. Although not yet fully modeled, this structure has already led to great insight into how P-Rex1 is regulated at the cell membrane, dissipating controversies that have existed in the P-Rex field for over a decade. By investigating P-Rex1 structure and regulation through a variety of complementary approaches, we hope to guide the future development of therapeutic molecules.
Compartmentalization of molecules is one of the key mechanisms of signaling regulation in live cells. Many components of the G protein signaling cascade exhibit preference towards distinct domains in the plasma membrane and change their subcellular localization upon activation. Many studies have attempted to determine domain localization of the members of the G protein signaling cascade both in the inactive and activated states. However, most studies done in live cells rely on high overexpression of proteins of interest. We set out to determine the spatial distribution of the G protein signaling components in clathrin-coated structures (CCSs) and caveolae at very low expression levels. To achieve this goal we used total internal reflection fluorescence microscopy (TIRF) and single-molecule imaging of CHO cells stably expressing SNAPf-tagged β2-adrenoceptors (SNAPf-β2AR). We achieved very low expression levels by imaging cells 2-3 hours after acute transfection of constructs driven by a CMV promoter or overnight transfection of constructs driven by a minimal promoter. Very low levels of expression allowed us to detect individual molecules of the proteins of interest without underlabeling the system. Clathrin light chain or caveolin1 were tagged with mNeonGreen fluorescent protein, SNAPf-β2AR was labeled with Snap-549 dye, and Halo-tagged subunits of Gs heterotrimers were labeled with Halo-JF646 dye. Our results show strong accumulation of SNAPf-β2AR and Halo-Arrestin3 after activation with the agonist isoproterenol in CCSs. In the same experiments neither Halo-Gαs nor Halo-Gγ2 subunits showed significant accumulation in CCSs after SNAPf-β2AR activation. Halo-Gαs and Halo-Gγ2 also did not show significant preference for caveolae either before or after activation. Our results show an absence of significant activated Gs protein compartmentalization in plasma membrane domains, including CCSs, arguing against coordinated internalization of G protein subunits through CCSs along with GPCRs after agonist stimulation.
Phospholipase Cε(PLCε) is among a family of phospholipase C (PLC) enzymes that hydrolyze phosphatidylinositol 4,5- bisphosphate (PIP2) into the second messengers diacylglycerol (DAG) and inositol 3,4,5 phosphate (IP3). PLCε enzymes can also hydrolyze phosphatidylinositol 4-phosphate (PI4P) at the perinuclear membrane and the Golgi. PLCε differ from other PLC enzymes as it contains an N-terminal CDC25 domain, which has guanine nucleotide exchange factor (GEF) activity towards small G proteins Rap1A,and two additional C-terminal Ras association (RA) domains. The best characterized pathway leading to PLCε activation is initiated by β-adrenergic receptors (βARs). Stimulation of this pathway results in the activation of Rap1A, which binds directly to the second C-terminal RA domain (RA2) of PLCε and translocates PLCε to the Golgi and increases its hydrolysis of PI4P. Prolonged activation of this pathway results in increased hypertrophic gene expression, leading to cardiac hypertrophy. There is minimal structural information for PLCε, and it is not known how Rap1A binding activates the enzyme. My goal is to determine the crystal structures of a catalytically active variant of PLCε and a complex between Rap1A and the isolated PLCε RA2. Ultimately, this work will lead to new approaches that could target PLCε in the treatment of cardiac hypertrophy.
Key steps in the canonical activation of heterotrimeric G proteins by G protein-coupled receptors (GPCRs) have been resolved at an atomic level. Non-canonical G protein signaling such as that triggered by non-receptor guanine-exchange modulators (GEMs), however, remains unresolved.
Here, we present a 2.1 Å crystal structure of Gαi in complex with a short G protein modulatory motif of the prototypical GEM, GIV (a.k.a Girdin). The structure reveals that GIV-GEM triggers GDP-exchange by binding to Gαi at an interface distinct from that employed by GPCRs and involving the hydrophobic cleft between the switch(Sw)-II and α3-helix. GIV directly engages the catalytic Glu (Q)204, thereby moving it away from SwI, and prevents Gai association with either Gbg or the canonical GoLoco motif-containing GDIs by both sterically occupying the key interaction residues and by inducing an incompatible conformation of SwII.
The structure also reveals how key phospho-modifications on GIV-GEM initiate or terminate signaling. Homology modeling revealed that these mechanisms are conserved among other GEMs.
Hydrogen-deuterium exchange experiments and molecular dynamics simulations demonstrate that binding-induced conformational changes in SwII allosterically propagate to and destabilize SwI, the helical domain, and the key hydrophobic cluster in the GTPase domain, all of which result in loosening GDP affinity and release acceleration. Notably, the structures of GPCR-bound Gαs and, more recently, GPCR-bound Gαi, all demonstrate that the GPCRs act via the same hydrophobic cluster in the G protein by inserting a hydrophobic residue from intracellular-loop 2 into this cleft. Thus, the simulations suggest that GEMs and GPCRs may accelerate GDP release via similar mechanisms, despite them occupying non-overlapping interfaces on Gαi.
Last, but not least, the MD simulations also highlight the roles of many Gαi residues whose contribution were not obvious from the structure; many of these residues were retrospectively found to play important roles in GDP binding (e.g. D173 and L175 whose mutations to alanine completely destabilizes Gαi and affects its ability to bind both GDP and GTP.
Because GEMs like GIV have emerged as key drivers of diverse pathophysiologic states, atomic-level insights presented here will enable the application of structure-based approaches to develop better strategies for selectively targeting the non-canonical G protein pathway.
With the recent explosion of available GPCR structures, a molecular understanding into the determinants of ligand bias is still lacking. Although key insights into the activation process of several prototypical GPCR structures have been presented, many of these structures are in complex with either an agonist or antagonist, which limits the interpretation into how biased activation may arise. Presented here are four crystal structures of the serotonin 5-HT2B receptor in complex with agonist, antagonist, and biased agonist, including a mutant crystal structure designed to convert an antagonist to an agonist. Together with extensive SAR and mutagenesis, these structures detail a binding pocket blueprint for not only how subtle differences in binding mode can lead to activation over inactivation, but also provide clues into how β-arrestin vs. G protein bias arises via ligand contact with sites distal from the orthosteric site. These studies not only indicate divergent mechanisms of allosteric versus orthosteric activation, but also indicate potential strategies to target putative allosteric sites for biased allosteric modulation for a new class of therapeutics aimed at mood disorders.
Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids to produce second messengers, including inositol-1,4,5-triphosphate (IP3) and diacylgycerol (DAG), which increase intracellular calcium and activate protein kinase C (PKC), respectively. PLCε contributes to cardiac hypertrophy and contractility, as well as to oncogenic and inflammatory signaling pathways downstream of receptor tyrosine kinases and G protein-coupled receptors. PLCε shares a conserved core with other PLC enzymes, but the roles of individual domains in activity and membrane binding have not been established. Using biochemical assays, small-angle X-ray scattering (SAXS), and electron microscopy (EM), we have found that the PLCε PH domain is necessary for basal lipase activity, but is dispensable for stability. Importantly, we provide the first structural insights into domain organization of PLCε, and reveal that the PH domain is conformationally heterogeneous in solution. Comparisons of the PLCε solution structure to that of the closely related PLCβ enzyme reveal that, in contrast to its crystal structure, the PLCβ PH domain is also mobile in solution. We also show the dynamic nature of the PH domain in these enzymes is functionally important, and may contribute to their regulation. These findings provide critical new insights into the solution structures of these signaling enzymes and their roles in cardiovascular disease and cancer.
Inflammation involves a plethora of mediators to promote either the inflammatory responses or the resolution of inflammation. Many of these mediators act on G protein-coupled receptors (GPCRs). Our research focuses on a group of non-chemokine chemoattractant GPCRs as close phylogenetic neighbors, including receptors for anaphylatoxins, formylpeptides, prostaglandin D2 (PGD2) and specialized pro-resolving lipid mediators. We aim to provide a comprehensive structural understanding of how this family of GPCRs recognizes such diverse peptide and lipid mediators, and further use the structural information to develop new agents for clinical investigation. With our recent progress, I would like to present a series of crystal structures of two representative members in this family, the C5aR (C5a receptor) for the anaphylatoxin peptide C5a and the CRTH2 (Chemokine Receptor homologous molecule expressed on Th2 lymphocytes) for the lipid molecule PGD2, bound to various antagonists. Those antagonists include two drug candidates that are currently in the late-stage clinical trials, avacopan for treating ANCA-associated vasculitis and fevipiprant for treating asthma. Our structures together with computational docking results reveal the orthosteric and allosteric action of chemically diverse C5aR antagonists, and the orthosteric action of two CRTH2 antagonists with a unique ligand binding-pocket. The structures of CRTH2 also suggest a novel mechanism for the recognition of lipid molecules by GPCRs. The future research goal is directed at elucidating the molecular mechanisms underlying eicosanoid lipid signaling in asthma.
By transmitting external signals to cell interior, guanine nucleotide-binding proteins (G-proteins) play a critical role in eukaryotic signal transduction. Prenylation is essential for both small monomeric and heterotrimeric G-proteins to interact with the plasma membrane (PM) and access their associated partners. Precursor lipids for prenylation, 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoids, are synthesized by the mevalonate (HMG-CoA reductase) pathway. Isoprenoids are intermediates of the mevalonate pathway, which also governs the hepatic cholesterol biosynthesis. Therefore, the rate-limiting enzyme of this pathway, HMG-CoA reductase is the primary target to numerous clinically-used cholesterol-lowering medications named statins. Since statins also inhibit prenylation of Ras proteins, they are used in chemotherapeutic drug regiments to attenuate cancer related motility. Although G protein gamma (Gγ) prenylation is essential for heterotrimeric G-protein function and these proteins regulate a majority of cellular functions and are implicated in multiple diseases, effects of statins on heterotrimeric G protein signaling is unclear. We show that, clinically-used statins extensively perturb PM localization of Gγ subtypes and thereby attenuate GPCR-G protein signaling. Interestingly, we also show that, disruption of membrane binding by statins is Gγ-subtype dependent where the farnesylation sensitive Gγ types are extensively sensitive to prenylation inhibition by statins. Further, variable efficiencies of prenylation inhibition of the same Gγ type by different statins were observed. For instance, out of the three statins examined, fluvastatin exhibited the highest as well as near-complete inhibition of Gγ farnesylation. Fluvastatin perturbed Gβγ mediated signaling including PIP3 production, PLC β induced calcium mobilization, and GRK mediated internalization of activated GPCRs. It also attenuated subsequent cell migration and invasion. Overall this study partly expands the potential of statins to be used as disease controllers by inhibiting signaling pathways driven by Gβγ while on the other hand this explains molecular mechanisms for some of the side effects reported with statins.
Introduction: Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion (> 35 replicates) in the amino-terminal region of the huntingtin protein (htt). Htt has the ability to bind to various transcription factors, such as the repressor of neuronal genes transcription (REST). However, these interactions are often disrupted in the presence of mutant huntingtin (mhtt). Metabotropic glutamate receptor 5 (mGluR5) signaling is crucial for controlling neuronal gene expression and mhtt interacts and alters mGluR5 signaling.
Objective: Investigate the influence of mGluR5 silencing on REST expression and its downstream signaling by genetic deletion of mGluR5 in the BACHD mouse model of HD, and by pharmacological inhibition of mGluR5 in a zQ175 knock-in mouse model using the mGluR5-specific negative allosteric modulator, CTEP.
Methods: We employed the BACHD transgenic mouse model of HD that expresses the mutated form of the htt, containing 97 glutamine repeats. mGluR5-/- mice were crossed with BACHD mice to generate BACHD mice lacking mGluR5 (BACHD/mGluR5-/-). We also employed 12-month-old heterozygous (Q175/+) and homozygous (Q175/Q175) knock-in mice (carrying ~188 CAG repeat expansions) treated with either vehicle or CTEP (2 mg/kg) for 12 weeks. qPCR and Western blot experiments were conducted on BACHD/mGluR5-/- at 2, 6 and 12 months of age and on zQ175 mice following CTEP treatment to explore the expression of REST and downstream signaling targets.
Results: We showed that both genetic (BACHD/mGluR5-/-) and pharmacological inhibition of mGluR5 (CTEP-treated Q175) lowered the expression of REST and increased the expression of SNAP25 and Synapsin compared to wild-type mice. However, Both BACHD and zQ175 mice did not exhibit a difference in REST expression compared to control mice. Interestingly, our results show that both mGluR5-/- and BACHD/mGluR5-/- display increased expression levels of REST target genes, indicating a dominant role for mGluR5 in regulating REST.
Conclusion: Our data provide evidence that mGluR5 regulates REST expression and its downstream signaling targets.
The delta opioid receptor (DOPr) has gained attention in recent years as agonists have been shown to promote analgesia and relieve depression. Additionally, evidence suggests that targeting this system results in less respiratory depression and abuse liability than the commonly targeted mu opioid receptor. Unfortunately, the clinical utility of DOPr agonists is limited as they lead to the rapid development of tolerance and many have proconvulsant properties. Evidence suggests that biased ligands may be capable of activating the downstream effectors associated with the positive effects of DOPr agonists, while mitigating the negative ones. BMS 986187 is a positive allosteric modulator (PAM) of the DOPr with apparent direct agonist properties, acting as an “ago-PAM”. Interacting with an allosteric site on DOPr, ago-PAM’s could promote biased receptor signaling leading to reduced on-target side effects. Based on preliminary data, this study set out to determine if BMS 986187 is acting as a biased ago-PAM. The results confirm that BMS 986187 is an ago-PAM at the DOPr with biased agonism favoring G protein activation over β-arrestin-mediated pathways, including receptor phosphorylation and internalization, resulting in reduced receptor desensitization. This is the first evidence of biased agonism mediated through direct binding to a unique allosteric site on the DOPr without occupancy of the orthosteric site. Our data suggests targeting the allosteric site on the DOPr, or indeed any GPCR, may be a novel way to promote signaling bias and thereby potentially produce a more targeted pharmacology.
Phospholipase C β (PLCβ) enzymes are peripheral membrane proteins that are required for normal cardiovascular function and whose dysregulation results in cardiovascular disease. PLCβ hydrolyzes phosphatidylinositiol-4,5-bisphosphate (PIP2) to produce the potent second messengers inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG), which increase intracellular Ca2+ and activate protein kinase C. PLCβ has low basal activity, but is activated downstream of G protein-coupled receptors (GPCRs) through direct interactions with the heterotrimeric G protein subunits Gαq and Gβγ. While G proteins stimulate activity, they are insufficient for full activation. This suggests that the membrane is required for full activation. However, the molecular basis for how the membrane and its properties contribute to PLCβ adsorption, activity, and Gαq-dependent activation are not well understood. We seek to understand how the membrane composition and its surface charge regulate adsorption of PLCβ to the membrane and promote interfacial activation, and the role of Gαq in this process. Using a model membrane system, we are applying an innovative combination of atomic force microscopy, Raman spectroscopy and biochemical assays to begin understanding how the membrane itself and Gαq regulate PLCβ activation. These studies provide the first structure-based approach to understanding how the cell membrane regulates the activity of this essential effector enzyme.
In the canonical model, the kappa opioid receptor (KOR) signals through Gαi/o proteins. However, the involvement of other Gα proteins to downstream signaling events has not been rigorously characterized. Using a BRET sensor, we previously found that when the KOR signals through Gαz, buprenorphine and samidorphan were full agonists. In contrast, they were both partial agonists when the KOR signaled through Gαi and Gαo (Bidlack, et al., JPET, 2018). Using two cell lines, HEK 293 and CHO, that endogenously express different Gα proteins, this study investigated how the milieu of Gα proteins influence the pharmacological properties of opioids. The differential Gα protein expression was characterized at mRNA and protein levels. Notably, HEK 293 cells expressed the pertussis toxin-insensitive Gαz, while CHO cells did not express Gαz. Changes in cAMP levels were measured in HEK 293 and CHO cells stably expressing the human KOR after administration of KOR agonists and antagonists. JDTic and naloxone, two classical antagonists, inhibited cAMP levels in HEK 293 cells, but did not in the CHOs. When Gαz was overexpressed in the CHO cells, JDTic behaved as a partial agonist. These results demonstrate that the individual Gα proteins have different effects on the pharmacological properties of opioid ligands resulting in functionally biased KOR signaling. (Supported by NIH T32GM068411 and the Margo Cleveland Fund.)
Major depressive disorder (MDD) is the most common mood disorder worldwide with a lifetime prevalence of ~15%. However, current FDA approved treatments are limited by a multi-week delayed onset of action and minimal efficacy in some patients. Preclinical evidence indicates delta-opioid receptor (DOR) agonists and kappa-opioid receptor (KOR) antagonists may produce rapid onset antidepressant effects and provide a novel depression target in treatment resistant patients [3H]-diprenorphine saturation binding experiments in membranes expressing the mu-opioid receptor (MOR); (KDm = 0.31 +/- 0.04); DOR (KDd = 1.1 nM +/- 0.16); and KOR (KOR KDk = 0.36 +/- 0.09) revealed non-selective affinity, as expected. In vitro [35S]GTPgS assays in the same cell lines revealed diprenorphine is a DOR and KOR partial agonist (EC50d = 4.1 nM +/- 2.0, EMAXd = 40 % +/- 5.0; EC50k = 0.71 nM +/- 0.21, EMAXk = 41 % +/- 5.3); it also has potent MOR antagonist against DAMGO (KB = 0.18 +/- 0.05). In vivo diprenorphine produced anti-depressive-like effects in the tail suspension test and the novelty-induced hypophagia test that were blocked by the DOR-selective antagonist naltrindole. While classical DOR agonists, such as SNC80, produce seizure activity, diprenorphine did not produce seizures and blocked SNC80 mediated seizures indicating that higher DOR efficacy is needed to produce seizures than anti-depressant-like effects. Alternatively, the DOR/KOR partial agonist and MOR antagonist profile of diprenorphine may mitigate against seizures. Future directions include the development of diprenorphine analogues to determine if modulating DOR/KOR agonist activity improves diprenorphine anti-depressant-like efficacy.
Parathyroid hormone (PTH) receptor (PTHR) is a family B G Protein Coupled Receptor (GPCR) that primarily couples to Gs/cAMP and Gq/Ca2+ signaling pathways and has a central role in regulating Ca2+ homeostasis and bone turnover. Structural studies of PTHR have proven particularly difficult due to instability of the receptor in the detergents. We have designed and optimized PTHR construct and purification procedure that yields stable and functional receptor at milligram quantities. The purified receptor, bound to a long-acting PTH analogue, forms nanocrystals at vapour-diffusion crystallization set-up, and makes a stable complex with wild-type Gs protein in presence of two stabilizing nanobodies (Nb35, and Nb37), as revealed by negative-stain transmission electron microscopy. We expect this complex to be suitable for cryo-EM structure determination.
One of the defining properties of the central nervous system is the ability of establishing precise synaptic contacts between neurons. This process involves trans-synaptic interactions between a host of cell-adhesion molecules, membrane receptors and secreted ligands that act in cooperation with the extracellular matrix to specify unique physiological properties of individual synapses. Traditionally, members of the G protein-coupled receptor (GPCR) family have been considered as powerful modulators of neurotransmission that shape properties of neuronal circuits. However, emerging proteomic studies increasingly point to their role in establishing trans-synaptic connections and interactions with the extracellular matrix. Such effects were primarily shown for the subfamily of adhesion GPCRs but the scope and extent of conservation across the GPCR superfamily is yet to be explored. Furthermore, the function and signaling properties of several GPCRs remain poorly understood, with many receptors still orphan of an endogenous ligand. Nonetheless, the identification of disease-causing mutations in patients and the analysis of transgenic models suggest that many orphan receptors have essential physiological roles and represent an attractive druggable target. Our progress in de-orphanizing these receptors and understanding their physiology has been slow, likely because of their unusual biology that may deviate from the traditional role of GPCRs as mediators of neurotransmitter signaling.
In this study, we identify major components of the extracellular matrix in the synaptic cleft - members of the Heparan Sulfate Proteoglycan (HSPG) family - as binding partners of the orphan GPCRs, GPR179 and GPR158, and demonstrate an essential role of these interactions in synaptic targeting. Using the mammalian retina as a model we provide evidence that the photoreceptor-released HSPG Pikachurin interacts with the orphan receptor GPR179 at the specific synapse between photoreceptors and ON-bipolar neurons. We further demonstrate that this organization dictates the post-synaptic targeting of the GAP complex by anchoring GPR179-RGS at the dendritic tips of downstream ON-bipolar neurons. Based on our observations we propose a model where Pikachurin, through its C-terminal EGF-like, Laminin G domain and HS side chains, acts as a bridge to connect the pre-synaptic Dystroglycan-Dystrophin complex at the photoreceptor axonal terminals with the extracellular domain of GPR179 on the post-synaptic site. Here GPR179 recruits cytoplasmic RGS proteins that are essential for specifying temporal properties of the synaptic transmission in response to light. Finally, using in vivo retina electroporation and several transgenic animal models, we confirm that ablation of single components of this trans-synaptic complex alters the post-synaptic recruitment of RGS proteins affecting the neurotransmission of photoreceptors.
Functional characterization of the GPCR interactome has focused predominantly on intracellular interacting-proteins, yet GPCRs are increasingly found in complex with extracellular proteins. Extracellular leucine rich repeat fibronectin type III domain containing 1 (ELFN1) was recently reported to physically anchor mGluR6 and mGluR7 across retinal and hippocampal synapses, respectively; however, the consequence and extent of trans-synaptic interactions on GPCR physiology and pharmacology had been unknown. We explored the functional implications of ELFN1-mGluR interactions by developing a novel transcellular GPCR signaling assay platform to monitor changes in mGluR activity in one cell following its exposure to separate ELFN1-containing cells. Using this platform, we found the first evidence of allosteric modulation of GPCR activity in trans; whereby ELFN1-expressing cells in co-culture altered both agonist-induced and constitutive receptor activity of group III mGluRs expressed in separate cell populations. We further adapted this platform for various readouts and determined transcellular ELFN1-mediated alterations of group III mGluR cAMP signaling was not a consequence of receptor membrane expression or desensitization, but instead a direct consequence of altered G protein activation as seen by BRET-based real-time kinetic assays. Furthermore, we have identified additional trans-synaptic binding partners of group III mGluRs with previously unknown function and demonstrate selective pharmacological consequence and relevance to neuropsychiatric disease. Our findings demonstrate that accepted principles of group III mGluR pharmacology are critically altered via extracellular interactions with endogenous trans-synaptic allosteric modulators, and disruption of these interactions may endow susceptibilities to neuropsychiatric disease.
There are 5 isoforms of Gβ and 12 isoforms of Gγ in humans, yet little is known about their distinct functions. While past research has focused on the Gα subunits of heterotrimeric G proteins, much less is known about Gβγ subunits. This project will explore the impact of different Gβγ isoforms on transcription in HEK 293 cells and in primary rat neonatal cardiac fibroblasts (RNCFs) in order to understand their unique roles in activating fibrotic signaling cascades. Based on previous work from our lab, we showed that Gβγ dimers are found at over 700 promoters in HEK 293 cells some of which are likely mediated by an interaction between Gβγ subunits and RNA polymerase II (RNAP II). This project will focus on adapting a proteomic screen in order to identify the interacting partners of the Gβγ isoforms at various stages in the transcription of individual genes. We will begin by setting up the screen to study specific gene loci under control conditions and following carbachol-stimulation of endogenous M3-mAChR in HEK 293 cells. We will use a technique called caspex to biotinylate proteins in proximity of a DNA sequence of interest. The APEX2 peroxidase is fused to dCas9, allowing the targeting of specific DNA sequences by guide RNAs and labelling of nearby proteins by biotinylation. Once labelled, these can be identified by mass spectrometry and confirmed by co-immunoprecipitation with FLAG-tagged Gβγ subunits. Following our experiments in HEK 293 cells, we will apply the screen in RNCFs and understand the proteomes at specific gene loci during the fibrotic response to Ang II with a view toward identifying how and when Gβγ subunits are recruited to target genes. Our results will establish a link between particular Gβγ isoforms and gene regulation through the generation of Gβγ dimer-specific interactomes.
G protein betagamma (Gβγ) interact with the plasma membrane (PM) through a prenyl group at the carboxy terminus (CT) of Gγ, which also strengthen the PM localization of Gαβγ heterotrimer as well. Gβγ subunits exhibit Gγ-type dependent unique PM affinities, thus signaling and cell behaviors, where PM affinity and signaling of Gβγ are proportional. However, it is not clear how Gβγ possess these unique PM affinities by having only two types of lipid anchors; either farnesyl or geranylgeranyl, on Gγ. Therefore, we explored the sequence properties of Gγ pre-prenylation region, which control the Gγ type-dependent differential PM affinity as well as Gβγ mediated cellular signaling. Results identified a key existence of hydrophobic residues in the pre-prenylation region (adjacent to the prenylated-cys in the CaaX motif) of Gγ types with the highest PM affinity. Further, we recognized the importance of them as a Gβγ signaling “on-off’’ switch, regardless of the type of prenylation on Gγ. Also, these pre-prenylation regions of Gγ are evolved to be significantly shorter than other prenylated proteins, however, still maintaining the G protein heterotrimer formation, GPCR-G protein interaction, and G protein signaling ability. Overall, this Gβγ-signaling “on-off” switch driven by the pre-prenylation region of Gγ has been identified and presented as a crucial regulator that dictates the signaling efficacy of Gβγ.
In 1999 Luttrell and colleagues provided compelling evidence that the β-Arrestin protein does more than silence activated G-protein coupled receptors 1. Indeed, β-Arrestin binds to over 300 other proteins in the cell and has its own role in signaling, a role we are still learning about 2. The ability of arrestin to signal appears to solve the riddle of how different ligands - acting at the same receptor - can produce different cellular responses: some produce more G-protein activation while others stimulate arrestin. This “biased agonism” is difficult to study, and often requires comparing the results of very different assays in different cells. Using an iterative process of design, build and test, we attempted to couple arrestin activation to changes in the fluorescence of mNeonGreen. Screening thousands of prototypes led to a fusion protein that produces a rapid decrease in fluorescence in response to GPCR activation. Since this green fluorescence is easily separated from the red fluorescence of the cADDis sensor for cAMP or the Downward DAG sensor for diacyl glycerol, both the β-Arrestin and G protein limbs of signaling can be measured simultaneously in living cells. In the case of the β2-adrenergic receptor - a class A GPCR 3 - a variety of agonists all stimulate the Gs limb, but only some produce a change in the β-Arrestin sensor: Isoproterenol and isoetharine produce rapid changes in red cADDis cAMP and green β-Arrestin sensors, while salbutamol, and salmeterol only produce a change in red cADDis. In the case of the Angiotensin II receptor - a class B GPCR - the red DAG and green β-Arrestin fluorescence signals reveal a different pattern of responses: Angiotensin II produces rapid DAG and β-Arrestin sensor responses, while the biased ligand SII produces no change in DAG and a much slower (10 fold) change in the β-Arrestin sensor. Experiments are underway with live cell imaging and perfusion to follow the entire time course of the responses.
The β2-adrenergic receptor (B2AR) was the first GPCR shown to extend the lifetime of endocytic domains it resides in. Subsequent studies with other GPCRs have shown a positive correlation between receptor endocytic lifetimes and mitogen activated protein kinase (MAPK) signaling. Although β-arrestins have been implicated in mediating this connection, the exact molecular mechanism regulating lifetime extension and connecting it to MAPK signaling remains unclear. We attempted to use B2AR as a model GPCR to determine this mechanism, relying on the requirement of its distal PDZ ligand for lifetime extension. We show that B2AR’s PDZ ligand is required for maximal MAPK activation downstream of isoproterenol. We then used shRNA to knockdown three known PDZ-dependent B2AR interactors (NHERF1, MAGI3, and SNX27) in HEK293 cells and evaluated receptor endocytic lifetimes and MAPK signaling. Surprisingly, we found that lifetime extension and MAPK signaling downstream of the receptor were regulated independently by distinct partners. Our findings dissociate lifetimes from MAPK signaling at B2AR and suggest that although endocytic lifetimes are a mechanism through which MAPK signaling can be modulated, this behavior is not conserved across all GPCRs.