Orphan GPCR GPRC5C Facilitates Angiotensin II-Induced Smooth Muscle Contraction.

BACKGROUND: GPCRs (G-protein-coupled receptors) play a central role in the regulation of smooth muscle cell (SMC) contractility, but the function of SMC-expressed orphan GPCR class C group 5 member C (GPRC5C) is unclear. The aim of this project is to define the role of GPRC5C in SMC in vitro and in vivo. METHODS: We studied the role of GPRC5C in the regulation of SMC contractility and differentiation in human and murine SMC in vitro, as well as in tamoxifen-inducible, SMC-specific GPRC5C knockout mice under basal conditions and in vascular disease in vivo. RESULTS: Mesenteric arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed ex vivo significantly reduced angiotensin II (Ang II)-dependent calcium mobilization and contraction, whereas responses to other relaxant or contractile factors were normal. In vitro, the knockdown of GPRC5C in human aortic SMC resulted in diminished Ang II-dependent inositol phosphate production and lower myosin light chain phosphorylation. In line with this, tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed reduced Ang II-induced arterial hypertension, and acute inactivation of GPRC5C was able to ameliorate established arterial hypertension. Mechanistically, we show that GPRC5C and the Ang II receptor AT1 dimerize, and knockdown of GPRC5C resulted in reduced binding of Ang II to AT1 receptors in HEK293 cells, human and murine SMC, and arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice. CONCLUSIONS: Our data show that GPRC5C regulates Ang II-dependent vascular contraction by facilitating AT1 receptor-ligand binding and signaling.

DP2 receptor activity sensor suited for antagonist screening and measurement of receptor dynamics in real-time.

The DP2 receptor is a G-protein coupled receptor involved in allergic inflammation and is the target of recently developed antagonists already being tested in clinics. To get insights into DP2 receptor dynamics and to study its pharmacology on the level of the receptor, we constructed a fluorescence resonance energy transfer-based conformation sensor. The sensor reflects the selectivity profile of the DP2 receptor-wt and is suited for screening of agonists and antagonists due to its robust response. Furthermore, the sensor enables the direct measurement of DP2 receptor dynamics in real-time and revealed markedly distinct on- and off-rates of prostaglandin D2 between DP2 and DP1 receptors, suggesting a different mechanism of ligand receptor interaction.

Differential interaction patterns of opioid analgesics with µ opioid receptors correlate with ligand-specific voltage sensitivity.

The µ opioid receptor (MOR) is the key target for analgesia, but the application of opioids is accompanied by several issues. There is a wide range of opioid analgesics, differing in their chemical structure and their properties of receptor activation and subsequent effects. A better understanding of ligand-receptor interactions and the resulting effects is important. Here, we calculated the respective binding poses for several opioids and analyzed interaction fingerprints between ligand and receptor. We further corroborated the interactions experimentally by cellular assays. As MOR was observed to display ligand-induced modulation of activity due to changes in membrane potential, we further analyzed the effects of voltage sensitivity on this receptor. Combining in silico and in vitro approaches, we defined discriminating interaction patterns responsible for ligand-specific voltage sensitivity and present new insights into their specific effects on activation of the MOR.

EP4 Receptor Conformation Sensor Suited for Ligand Screening and Imaging of Extracellular Prostaglandins.

Prostaglandins are important lipid mediators with a wide range of functions in the human body. They act mainly via plasma membrane localized prostaglandin receptors, which belong to the G-protein coupled receptor class. Due to their localized formation and short lifetime, it is important to be able to measure the distribution and abundance of prostaglandins in time and/or space. In this study, we present a Foerster resonance energy transfer (FRET)-based conformation sensor of the human prostaglandin E receptor subtype 4 (EP4 receptor), which was capable of detecting prostaglandin E2 (PGE2)-induced receptor activation in the low nanomolar range with a good signal-to-noise ratio. The sensor retained the typical selectivity for PGE2 among arachidonic acid products. Human embryonic kidney cells stably expressing the sensor did not produce detectable amounts of prostaglandins making them suitable for a coculture approach allowing us, over time, to detect prostaglandin formation in Madin-Darby canine kidney cells and primary mouse macrophages. Furthermore, the EP4 receptor sensor proved to be suited to detect experimentally generated PGE2 gradients by means of FRET-microscopy, indicating the potential to measure gradients of PGE2 within tissues. In addition to FRET-based imaging of prostanoid release, the sensor allowed not only for determination of PGE2 concentrations, but also proved to be capable of measuring ligand binding kinetics. The good signal-to-noise ratio at a commercial plate reader and the ability to directly determine ligand efficacy shows the obvious potential of this sensor interest for screening and characterization of novel ligands of the pharmacologically important human EP4 receptor. SIGNIFICANCE STATEMENT: The authors present a biosensor based on the prostaglandin E receptor subtype 4, which is well suited to measure extracellular prostaglandin E2 (PGE2) concentration with high temporal and spatial resolution. It can be used for the imaging of PGE2 levels and gradients by means of Foerster resonance energy transfer microscopy, and for determining PGE2 release of primary cells as well as for screening purposes in a plate reader setting.

Interaction kinetics between p115-RhoGEF and Gα13 are determined by unique molecular interactions affecting agonist sensitivity.

The three RH-RhoGEFs (Guanine nucleotide exchange factors) p115-RhoGEF, LARG (leukemia-associated RhoGEF) and PDZ-RhoGEF link G-protein coupled receptors (GPCRs) with RhoA signaling through activation of Gα12/13. In order to find functional differences in signaling between the different RH-RhoGEFs we examined their interaction with Gα13 in high spatial and temporal resolution, utilizing a FRET-based single cell assay. We found that p115-RhoGEF interacts significantly shorter with Gα13 than LARG and PDZ-RhoGEF, while narrowing the structural basis for these differences down to a single amino acid in the rgRGS domain of p115-RhoGEF. The mutation of this amino acid led to an increased interaction time with Gα13 and an enhanced agonist sensitivity, comparable to LARG, while mutating the corresponding amino acid in Gα13 the same effect could be achieved. While the rgRGS domains of RH-RhoGEFs showed GAP (GTPase-activating protein) activity towards Gα13 in vitro, our approach suggests higher GAP activity of p115-RhoGEF in intact cells.

Differential effects of glucose-dependent insulinotropic polypeptide receptor/glucagon-like peptide-1 receptor heteromerization on cell signaling when expressed in HEK-293 cells.

The incretin hormones: glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are important regulators of many aspects of metabolism including insulin secretion. Their receptors (GIPR and GLP-1R) are closely related members of the secretin class of G-protein-coupled receptors. As both receptors are expressed on pancreatic ß-cells there is at least the hypothetical possibility that they may form heteromers. In the present study, we investigated GIPR/GLP-1R heteromerization and the impact of GIPR on GLP-1R-mediated signaling and vice versa in HEK-293 cells. Real-time fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) saturation experiments confirm that GLP-1R and GIPR form heteromers. Stimulation with 1 µM GLP-1 caused an increase in both FRET and BRET ratio, whereas stimulation with 1 µM GIP caused a decrease. The only other ligand tested to cause a significant change in BRET signal was the GLP-1 metabolite, GLP-1 (9-36). GIPR expression had no significant effect on mini-Gs recruitment to GLP-1R but significantly inhibited GLP-1 stimulated mini-Gq and arrestin recruitment. In contrast, the presence of GLP-1R improved GIP stimulated mini-Gs and mini-Gq recruitment to GIPR. These data support the hypothesis that GIPR and GLP-1R form heteromers with differential consequences on cell signaling.

The pocketome of G-protein-coupled receptors reveals previously untargeted allosteric sites.

G-protein-coupled receptors do not only feature the orthosteric pockets, where most endogenous agonists bind, but also a multitude of other allosteric pockets that have come into the focus as potential binding sites for synthetic modulators. Here, to better characterise such pockets, we investigate 557 GPCR structures by exhaustively docking small molecular probes in silico and converting the ensemble of binding locations to pocket-defining volumes. Our analysis confirms all previously identified pockets and reveals nine previously untargeted sites. In order to test for the feasibility of functional modulation of receptors through binding of a ligand to such sites, we mutate residues in two sites, in two model receptors, the muscarinic acetylcholine receptor M3 and ß2-adrenergic receptor. Moreover, we analyse the correlation of inter-residue contacts with the activation states of receptors and show that contact patterns closely correlating with activation indeed coincide with these sites.

Residency time of agonists does not affect the stability of GPCR-arrestin complexes.

BACKGROUND AND PURPOSE: The interaction of arrestins with G-protein coupled receptors (GPCRs) desensitizes agonist-dependent receptor responses and often leads to receptor internalization. GPCRs that internalize without arrestin have been classified as "class A" GPCRs whereas "class B" GPCRs co-internalize with arrestin into endosomes. The interaction of arrestins with GPCRs requires both agonist activation and receptor phosphorylation. Here, we ask the question whether agonists with very slow off-rates can cause the formation of particularly stable receptor-arrestin complexes. EXPERIMENTAL APPROACH: The stability of GPCR-arrestin-3 complexes at two class A GPCRs, the ß2 -adrenoceptor and the µ opioid receptor, was assessed using two different techniques, fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) employing several ligands with very different off-rates. Arrestin trafficking was determined by confocal microscopy. KEY RESULTS: Upon agonist washout, GPCR-arrestin-3 complexes showed markedly different dissociation rates in single-cell FRET experiments. In FRAP experiments, however, all full agonists led to the formation of receptor-arrestin complexes of identical stability whereas the complex between the µ receptor and arrestin-3 induced by the partial agonist morphine was less stable. Agonists with very slow off-rates could not mediate the co-internalization of arrestin-3 with class A GPCRs into endosomes. CONCLUSIONS AND IMPLICATIONS: Agonist off-rates do not affect the stability of GPCR-arrestin complexes but phosphorylation patterns do. Our results imply that orthosteric agonists are not able to pharmacologically convert class A into class B GPCRs.

Disentangling bias between Gq, GRK2, and arrestin3 recruitment to the M3 muscarinic acetylcholine receptor.

G protein-coupled receptors (GPCRs) transmit extracellular signals to the inside by activation of intracellular effector proteins. Different agonists can promote differential receptor-induced signaling responses - termed bias - potentially by eliciting different levels of recruitment of effector proteins. As activation and recruitment of effector proteins might influence each other, thorough analysis of bias is difficult. Here, we compared the efficacy of seven agonists to induce G protein, G protein-coupled receptor kinase 2 (GRK2), as well as arrestin3 binding to the muscarinic acetylcholine receptor M3 by utilizing FRET-based assays. In order to avoid interference between these interactions, we studied GRK2 binding in the presence of inhibitors of Gi and Gq proteins and analyzed arrestin3 binding to prestimulated M3 receptors to avoid differences in receptor phosphorylation influencing arrestin recruitment. We measured substantial differences in the agonist efficacies to induce M3R-arrestin3 versus M3R-GRK2 interaction. However, the rank order of the agonists for G protein- and GRK2-M3R interaction was the same, suggesting that G protein and GRK2 binding to M3R requires similar receptor conformations, whereas requirements for arrestin3 binding to M3R are distinct.

Structures in G proteins important for subtype selective receptor binding and subsequent activation.

G protein-coupled receptors (GPCRs) selectively couple to specific heterotrimeric G proteins comprised of four subfamilies in order to induce appropriate physiological responses. However, structural determinants in Gα subunits responsible for selective recognition by approximately 800 human GPCRs have remained elusive. Here, we directly compare the influence of subtype-specific Gα structures on the stability of GPCR-G protein complexes and the activation by two Gq-coupled receptors. We used FRET-assays designed to distinguish multiple Go and Gq-based Gα chimeras in their ability to be selectively bound and activated by muscarinic M3 and histaminic H1 receptors. We identify the N-terminus including the αN/ß1-hinge, the ß2/ß3-loop and the α5 helix of Gα to be key selectivity determinants which differ in their impact on selective binding to GPCRs and subsequent activation depending on the specific receptor. Altogether, these findings provide new insights into the molecular basis of G protein-coupling selectivity even beyond the Gα C-terminus.

Pharmacological Characterization of the Stick Insect Carausius morosus Allatostatin-C Receptor with Its Endogenous Agonist.

G protein-coupled receptors (GPCRs) play a pivotal role in regulating key physiological events in all animal species. Recent advances in collective analysis of genes and proteins revealed numerous potential neuropeptides and GPCRs from insect species, allowing for the characterization of peptide-receptor pairs. In this work, we used fluorescence resonance energy transfer (FRET)-based genetically encoded biosensors in intact mammalian cells to study the pharmacological features of the cognate GPCR of the type-C allatostatin (AST-C) peptide from the stick insect, Carausius morosus. Analysis of multiple downstream pathways revealed that AST-C can activate the human Gi2 protein, and not Gs or Gq, through AST-C receptor (AlstRC). Activated AlstRC recruits ß-arrestin2 independent of the Gi protein but stimulates ERK phosphorylation in a Gi protein-dependent manner. Identification of Gαi-, arrestin-, and GRK-like transcripts from C. morosus revealed high evolutionary conservation at the G protein level, while ß-arrestins and GRKs displayed less conservation. In conclusion, our study provides experimental and homology-based evidence on the functionality of vertebrate G proteins and downstream signaling biosensors to characterize early signaling steps of an insect GPCR. These results may serve as a scaffold for developing assays to characterize pharmacological and structural aspects of other insect GPCRs and can be used in deorphanization and pesticide studies.

The Effect of Cell Surface Expression and Linker Sequence on the Recruitment of Arrestin to the GIP Receptor.

The glucose-dependent insulinotropic polypeptide (GIP) and the glucagon-like peptide-1 (GLP-1) receptor are important targets in the treatment of both type 2 diabetes mellitus (T2DM) and obesity. Originally identified for their role in desensitization, internalization and recycling of G protein-coupled receptors (GPCRs), arrestins have since been shown to act as scaffolding proteins that allow GPCRs to signal in a G protein-independent manner. While GLP-1R has been reported to interact with arrestins, this aspect of cell signaling remains controversial for GIPR. Using a (FRET)-based assay we have previously shown that yellow fluorescent protein (YFP)-labeled GIPR does not recruit arrestin. This GIPR-YFP construct contained a 10 amino acid linker between the receptor and a XbaI restriction site upstream of the YFP. This linker was not present in the modified GIPR-SYFP2 used in subsequent FRET and bioluminescence resonance energy transfer (BRET) assays. However, its removal results in the introduction of a serine residue adjacent to the end of GIPR's C-terminal tail which could potentially be a phosphorylation site. The resulting receptor was indeed able to recruit arrestin. To find out whether the serine/arginine (SR) coded by the XbaI site was indeed the source of the problem, it was substituted with glycine/glycine (GG) by site-directed mutagenesis. This substitution abolished arrestin recruitment in the BRET assay but only significantly reduced it in the FRET assay. In addition, we show that the presence of a N-terminal FLAG epitope and influenza hemagglutinin signal peptide were also required to detect arrestin recruitment to the GIPR, most likely by increasing receptor cell surface expression. These results demonstrate how arrestin recruitment assay configuration can dramatically alter the result. This becomes relevant when drug discovery programs aim to identify ligands with "biased agonist" properties.

Voltage modulates the effect of µ-receptor activation in a ligand-dependent manner.

BACKGROUND AND PURPOSE: Various GPCRs have been described as being modulated in a voltage-dependent manner. Opioid analgesics act via activation of µ receptors in various neurons. As neurons are exposed to large changes in membrane potential, we were interested in studying the effects of depolarization on µ receptor signalling. EXPERIMENTAL APPROACH: We investigated potential voltage sensitivity of µ receptors in heterologous expression systems (HEK293T cells) using electrophysiology in combination with Förster resonance energy transfer-based assays. Depolarization-induced changes in signalling were also tested in physiological rat tissue containing locus coeruleus neurons. We applied depolarization steps across the physiological range of membrane potentials. KEY RESULTS: Studying µ receptor function and signalling in cells, we discovered that morphine-induced signalling was strongly dependent on the membrane potential (VM ). This became apparent at the level of G-protein activation, G-protein coupled inwardly rectifying potassium channel (Kir 3.X) currents and binding of GPCR kinases and arrestin3 to µ receptors by a robust increase in signalling upon membrane depolarization. The pronounced voltage sensitivity of morphine-induced µ receptor activation was also observed at the level of Kir 3.X currents in rat locus coeruleus neurons. The efficacy of peptide ligands to activate µ receptors was not (Met-enkephalin) or only moderately ([D-Ala2 , N-Me-Phe4 , Gly5 -ol]-enkephalin) enhanced upon depolarization. In contrast, depolarization reduced the ability of the analgesic fentanyl to activate µ receptors. CONCLUSION AND IMPLICATIONS: Our results indicate a strong ligand-dependent modulation of µ receptor activity by the membrane potential, suggesting preferential activity of morphine in neurons with high neuronal activity.

Voltage Dependence of Prostanoid Receptors.

G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors and serve as signal mediators to transduce information from extracellular signals such as neurotransmitters, hormones, or drugs to cellular responses. They are exposed to the strong electrical field of the plasma membrane. In the last decade voltage modulation of ligand-induced GPCR activity has been reported for several GPCRs. Using Foerster resonance energy transfer-based biosensors in patch clamp experiments, we discovered a robust voltage dependence of the thromboxane receptor (TP receptor) on the receptor level as well as on downstream signaling. TP receptor activity doubled upon depolarization from -90 to +60 mV in the presence of U46619, a stable analog of prostaglandin H2 Half-maximal effective potential (V0.5) determined for TP receptor was -46 mV, which is within the physiologic range. We identified that depolarization affected the agonist affinity for the TP receptor. Depolarization enhanced responses of several structural analogs of U46619 with modifications to a similar extent all around the molecule, indicating that voltage modulates the general conformation of TP receptor. By means of site direct mutagenesis, we identified TP receptor R2957.40, which showed alteration of voltage sensitivity of TP receptor upon mutation. Voltage sensitivity was not limited to TP receptor because prostaglandin F receptor activated with U46619 and prostaglandin E2 receptor subtype 3 activated with iloprost showed a similar reaction to depolarization as TP receptor. However, prostacyclin receptor activated with iloprost showed no detectable voltage dependence. SIGNIFICANCE STATEMENT: Prostanoids mediate many of their physiological effects via transmembrane receptors expressed in the plasma membrane of excitable cells. We found that agonist-mediated activation of prostaglandin F receptors and prostaglandin E2 receptors as well as thromboxane receptors are activated upon depolarization, whereas prostacyclin receptors are not. The voltage-induced modulation of thromboxane receptor activity was observed on the level of receptor conformation and downstream signaling. The range of voltage dependence was restricted by R2957.40 in the agonist-binding pocket.

Orphan G Protein-Coupled Receptor GPRC5B Controls Smooth Muscle Contractility and Differentiation by Inhibiting Prostacyclin Receptor Signaling.

BACKGROUND: G protein-coupled receptors are important regulators of contractility and differentiation in vascular smooth muscle cells (SMCs), but the specific function of SMC-expressed orphan G protein-coupled receptor class C group 5 member B (GPRC5B) is unclear. METHODS: We studied the role of GPRC5B in the regulation of contractility and dedifferentiation in human and murine SMCs in vitro and in iSM-Gprc5b-KO (tamoxifen-inducible, SMC-specific knockout) mice under conditions of arterial hypertension and atherosclerosis in vivo. RESULTS: Mesenteric arteries from SMC-specific Gprc5b-KOs showed ex vivo significantly enhanced prostacyclin receptor (IP)-dependent relaxation, whereas responses to other relaxant or contractile factors were normal. In vitro, knockdown of GPRC5B in human aortic SMCs resulted in increased IP-dependent cAMP production and consecutive facilitation of SMC relaxation. In line with this facilitation of IP-mediated relaxation, iSM-Gprc5b-KO mice were protected from arterial hypertension, and this protective effect was abrogated by IP antagonists. Mechanistically, we show that knockdown of GPRC5B increased the membrane localization of IP both in vitro and in vivo and that GPRC5B, but not other G protein-coupled receptors, physically interacts with IP. Last, we show that enhanced IP signaling in GPRC5B-deficient SMCs not only facilitates relaxation but also prevents dedifferentiation during atherosclerosis development, resulting in reduced plaque load and increased differentiation of SMCs in the fibrous cap. CONCLUSIONS: Taken together, our data show that GPRC5B regulates vascular SMC tone and differentiation by negatively regulating IP signaling.

The N-termini of GRK2 and GRK3 simulate the stimulating effects of RKIP on ß-adrenoceptors.

The Raf kinase inhibitor protein (RKIP) activates ß-adrenoceptors (ß-AR) and thereby induces a well-tolerated cardiac contractility and prevents heart failure in mice. Different to RKIP-mediated ß-AR activation, chronic activation of ß-AR by catecholamines was shown to be detrimental for the heart. RKIP is an endogenous inhibitor of G protein coupled receptor kinase 2 (GRK2); it binds GRK2 and thereby inhibits GRK2 mediated ß-AR phosphorylation and desensitization. Here, we evaluate RKIP-mediated effects on ß-AR to explore new strategies for ß-AR modulation. Co-immunoprecipitation assays and pull-down assays revealed subtype specificity of RKIP for the cardiac GRK isoforms GRK2 and GRK3 - not GRK5 - as well as several RKIP binding sites within their N-termini (GRK21-185 and GRK31-185). Overexpression of these N-termini prevented ß2-AR phosphorylation and internalization, subsequently increased receptor signaling in HEK293 cells and cardiomyocyte contractility. Co-immunoprecipitation assays of ß2-AR with these N-terminal GRK fragments revealed a direct interaction suggesting a steric interference of the fragments with the functional GRK-receptor interaction. Altogether, N-termini of GRK2 and GRK3 efficiently simulate RKIP effects on ß-AR signaling in HEK293 cells and in cardiomyocytes by their binding to ß2-AR and, thus, provide important insights for the development of new strategies to modulate ß2-AR signaling.

Comparative Docking to Distinct G Protein-Coupled Receptor Conformations Exclusively Yields Ligands with Agonist Efficacy.

G protein-coupled receptors exist in a whole spectrum of conformations that are stabilized by the binding of ligands with different efficacy or intracellular effector proteins. Here, we investigate whether three-dimensional structures of receptor conformations in different states of activation can be used to enrich ligands with agonist behavior in prospective docking calculations. We focused on the ß 2-adrenergic receptor, as it is currently the receptor with the highest number of active-state crystal structures. Comparative docking calculations to distinct conformations of the receptor were used for the in silico prediction of ligands with agonist efficacy. The pharmacology of molecules selected based on these predictions was characterized experimentally, resulting in a hit rate of 37% ligands, all of which were agonists. The ligands furthermore contain a pyrazole moiety that has previously not been described for ß 2-adrenergic receptor ligands, and one of them shows an intrinsic efficacy comparable to salbutamol. SIGNIFICANCE STATEMENT: Structure-based ligand design for G protein-coupled receptors crucially depends on receptor conformation and, hence, their activation state. We explored the influence of using multiple active-conformation X-ray structures on the hit rate of docking calculations to find novel agonists, and how to predict the most fruitful strategy to apply. The results suggest that aggregating the ranks of molecules across docking calculations to more than one active-state structure exclusively yields agonists.

Direct targeting of Gαq and Gα11 oncoproteins in cancer cells.

Somatic gain-of-function mutations of GNAQ and GNA11, which encode α subunits of heterotrimeric Gαq/11 proteins, occur in about 85% of cases of uveal melanoma (UM), the most common cancer of the adult eye. Molecular therapies to directly target these oncoproteins are lacking, and current treatment options rely on radiation, surgery, or inhibition of effector molecules downstream of these G proteins. A hallmark feature of oncogenic Gαq/11 proteins is their reduced intrinsic rate of hydrolysis of guanosine triphosphate (GTP), which results in their accumulation in the GTP-bound, active state. Here, we report that the cyclic depsipeptide FR900359 (FR) directly interacted with GTPase-deficient Gαq/11 proteins and preferentially inhibited mitogenic ERK signaling rather than canonical phospholipase Cß (PLCß) signaling driven by these oncogenes. Thereby, FR suppressed the proliferation of melanoma cells in culture and inhibited the growth of Gαq-driven UM mouse xenografts in vivo. In contrast, FR did not affect tumor growth when xenografts carried mutated B-RafV600E as the oncogenic driver. Because FR enabled suppression of malignant traits in cancer cells that are driven by activating mutations at codon 209 in Gαq/11 proteins, we envision that similar approaches could be taken to blunt the signaling of non-Gαq/11 G proteins.

Multisite phosphorylation is required for sustained interaction with GRKs and arrestins during rapid µ-opioid receptor desensitization.

G protein receptor kinases (GRKs) and ß-arrestins are key regulators of µ-opioid receptor (MOR) signaling and trafficking. We have previously shown that high-efficacy opioids such as DAMGO stimulate a GRK2/3-mediated multisite phosphorylation of conserved C-terminal tail serine and threonine residues, which facilitates internalization of the receptor. In contrast, morphine-induced phosphorylation of MOR is limited to Ser375 and is not sufficient to drive substantial receptor internalization. We report how specific multisite phosphorylation controlled the dynamics of GRK and ß-arrestin interactions with MOR and show how such phosphorylation mediated receptor desensitization. We showed that GRK2/3 was recruited more quickly than was ß-arrestin to a DAMGO-activated MOR. ß-Arrestin recruitment required GRK2 activity and MOR phosphorylation, but GRK recruitment also depended on the phosphorylation sites in the C-terminal tail, specifically four serine and threonine residues within the 370TREHPSTANT379 motif. Our results also suggested that other residues outside this motif participated in the initial and transient recruitment of GRK and ß-arrestins. We identified two components of high-efficacy agonist desensitization of MOR: a sustained component, which required GRK2-mediated phosphorylation and a potential soluble factor, and a rapid component, which was likely mediated by GRK2 but independent of receptor phosphorylation. Elucidating these complex receptor-effector interactions represents an important step toward a mechanistic understanding of MOR desensitization that leads to the development of tolerance and dependence.

Lifetime of muscarinic receptor-G-protein complexes determines coupling efficiency and G-protein subtype selectivity.

G-protein-coupled receptors (GPCRs) are essential for the detection of extracellular stimuli by cells and transfer the encoded information via the activation of functionally distinct subsets of heterotrimeric G proteins into intracellular signals. Despite enormous achievements toward understanding GPCR structures, major aspects of the GPCR-G-protein selectivity mechanism remain unresolved. As this can be attributed to the lack of suitable and broadly applicable assays, we set out to develop a quantitative FRET-based assay to study kinetics and affinities of G protein binding to activated GPCRs in membranes of permeabilized cells in the absence of nucleotides. We measured the association and dissociation kinetics of agonist-induced binding of Gi/o, Gq/11, Gs, and G12/13 proteins to muscarinic M1, M2, and M3 receptors in the absence of nucleotides between fluorescently labeled G proteins and receptors expressed in mammalian cells. Our results show a strong quantitative correlation between not the on-rates of G-protein-M3-R interactions but rather the affinities of Gq and Go proteins to M3-Rs, their GPCR-G-protein lifetime and their coupling efficiencies determined in intact cells, suggesting that the G-protein subtype-specific affinity to the activated receptor in the absence of nucleotides is, in fact, a major determinant of the coupling efficiency. Our broadly applicable FRET-based assay represents a fast and reliable method to quantify the intrinsic affinity and relative coupling selectivity of GPCRs toward all G-protein subtypes.