Deorphanization of G Protein Coupled Receptors: A Historical Perspective.

Counting over 800 members, G protein coupled receptors (GPCRs) form the largest family of membrane receptors encoded in the human genome. Since the discovery of G proteins and GPCRs in the late 1970s and early 1980s, a significant portion of the GPCR research has been focused on identifying ligand/receptor pairs in parallel to studies related to their signaling properties. Despite significant advancements, about a fourth of the ∼400 nonodorant GPCRs are still considered orphan because their natural or endogenous ligands have yet to be identified. We should consider that every GPCR was once an orphan and that endogenous ligands have often been associated with biologic effects without a complete understanding of the molecular identity of their target receptors. Within this framework, this review offers a historical perspective on deorphanization processes for representative GPCRs, including the ghrelin receptor, γ aminobutyric acid B receptor, apelin receptor, cannabinoid receptors, and GPR15. It explores three main scenarios encountered in deorphanization efforts and discusses key questions and methodologies employed in elucidating ligand-receptor interactions, providing insights for future research endeavors. SIGNIFICANCE STATEMENT: Understanding how scientists have historically approached the issue of GPCR deorphanization and pairing of biologically active ligands with their cognate receptors are relevant topics in pharmacology. In fact, the biology of each GPCR, including its pathophysiological involvement, has often been uncovered only after its deorphanization, illuminating druggable targets for various diseases. Furthermore, uncovered endogenous ligands have therapeutic value as many ligands-or derivates thereof-are developed into drugs.

Rationale and design of the RAPID-WATER-FLOW trial: Radiolabeled perfusion to identify coronary artery disease using water to evaluate responses of myocardial FLOW.

OBJECTIVES: The objective of this study was to determine the diagnostic performance of 15O-water positron emission tomography (PET) myocardial perfusion imaging to detect coronary artery disease (CAD) using the truth-standard of invasive coronary angiography (ICA) with fractional flow reserve (FFR) or instantaneous wave-Free Ratio (iFR) or coronary computed tomography angiogram (CCTA). BACKGROUND: 15O-water has a very high first-pass extraction that allows accurate quantification of myocardial blood flow and detection of flow-limiting CAD. However, the need for an on-site cyclotron and lack of automated production at the point of care and relatively complex image analysis protocol has limited its clinical use to date. METHODS: The RAPID WATER FLOW study is an open-label, multicenter, prospective investigation of the accuracy of 15O-water PET to detect obstructive angiographic and physiologically significant stenosis in patients with suspected CAD. The study will include the use of an automated system for producing, dosing, and injecting 15O-water and enrolling approximately 215 individuals with suspected CAD at approximately 10 study sites in North America and Europe. The primary endpoint of the study is the diagnostic sensitivity and specificity of the 15O-water PET study using the truth-standard of ICA with FFR or iFR to determine flow-limiting stenosis, or CCTA to rule out CAD and incorporating a quantitative analytic platform developed for the 15O-water PET acquisitions. Sensitivity and specificity are to be considered positive if the lower bound of the 95% confidence interval is superior to the threshold of 60% for both, consistent with prior registration studies. Subgroup analyses include assessments of diagnostic sensitivity, specificity, and accuracy in female, obese, and diabetic individuals, as well as in those with multivessel disease. All enrolled individuals will be followed for adverse and serious adverse events for up to 32 hours after the index PET scan. The study will have >90% power (one-sided test, α = 0.025) to test the hypothesis that sensitivity and specificity of 15O-water PET are both >60%. CONCLUSIONS: The RAPID WATER FLOW study is a prospective, multicenter study to determine the diagnostic sensitivity and specificity of 15O-water PET as compared to ICA with FFR/iFR or CCTA. This study will introduce several novel aspects to imaging registration studies, including a more relevant truth standard incorporating invasive physiologic indexes, coronary CTA to qualify normal individuals for eligibility, and a more quantitative approach to image analysis than has been done in prior pivotal studies. CLINICAL TRIAL REGISTRATION INFORMATION: Clinical-Trials.gov (#NCT05134012).

Gα Protein Signaling Bias at Serotonin 1A Receptor.

Serotonin 1A receptor (5-HT1AR) is a clinically relevant target because of its involvement in several central and peripheral functions, including sleep, temperature homeostasis, processing of emotions, and response to stress. As a G protein coupled receptor (GPCR) activating numerous Gα i/o/z family members, 5-HT1AR can potentially modulate multiple intracellular signaling pathways in response to different therapeutics. Here, we applied a cell-based bioluminescence resonance energy transfer assay to quantify how ten structurally diverse 5-HT1AR agonists exert biased signaling by differentially stimulating Gα i/o/z family members. Our concentration-response analysis of the activation of each Gα i/o/z protein revealed unique potency and efficacy profiles of selected agonists when compared with the reference 5-hydroxytryptamine, serotonin. Overall, our analysis of signaling bias identified groups of ligands sharing comparable G protein activation selectivity and also drugs with unique selectivity profiles. We observed, for example, a strong bias of F-15599 toward the activation of Gα i3 that was unique among the agonists tested: we found a biased factor of +2.19 when comparing the activation of Gα i3 versus Gα i2 by F-15599, while it was -0.29 for 8-hydroxy-2-(di-n-propylamino) tetralin. Similarly, vortioxetine showed a biased factor of +1.06 for Gα z versus Gα oA, while it was -1.38 for vilazodone. Considering that alternative signaling pathways are regulated downstream of each Gα protein, our data suggest that the unique pharmacological properties of the tested agonists could result in multiple unrelated cellular outcomes. Further investigation is needed to reveal how this type of ligand bias could affect cellular responses and to illuminate the molecular mechanisms underlying therapeutic profile and side effects of each drug. SIGNIFICANCE STATEMENT: Serotonin 1a receptor (5-HT1AR) activates several members of the Gi/o/z protein family. Here, we examined ten structurally diverse and clinically relevant agonists acting on 5-HT1AR and identified distinctive bias patterns among G proteins. Considering the diversity of their intracellular effectors and signaling properties, this data reveal novel mechanisms underlying both therapeutic and undesirable effects.

Probing the orphan receptors: Tools and directions.

The endogenous ligands activating a large fraction of the G Protein Coupled Receptor (GPCR) family members have yet to be identified. These receptors are commonly labeled as orphans (oGPCRs), and because of the absence of available pharmacological tools they are currently understudied. Nonetheless, genome wide association studies, together with research using animal models identified many physiological functions regulated by oGPCRs. Similarly, mutations in some oGPCRs have been associated with rare genetic disorders or with an increased risk of developing pathologies. The once underestimated pharmacological potential of targeting oGPCRs is increasingly being exploited by the development of novel tools to understand their biology and by drug discovery endeavors aimed at identifying new modulators of their activity. Here, we summarize recent advancements in the field of oGPCRs and future directions.

Comprehensive Spatial Profile of the Orphan G Protein Coupled Receptor GPRC5B Expression in Mouse Brain.

Orphan G Protein Coupled Receptors (GPCRs) are GPCRs whose endogenous ligands are unknown or still debated. Due to the lack of pharmacological modulators, the physiological function of orphan GPCRs is understudied. However, relevant physiological roles associated with orphan GPCRs have been revealed by analysis of animal models and genome wide association studies illuminating an untapped potential for drug discovery. G Protein Coupled Receptor class C Group 5 Member B (GPRC5B) is among the most expressed GPCRs in the central nervous system. Thus, the expression profiling of GPRC5B is an essential step toward understanding GPRC5B function in health and disease. In this study, we generated new GPRC5B polyclonal antibodies and investigated the expression levels of GPRC5B across different organs and brain regions. We identified high levels of GPRC5B glycosylation both in transfected cells and in mouse brain. Moreover, in situ hybridization imaging analysis indicated that Gprc5b was expressed at the highest level in olfactory bulb, hippocampus, cerebellum, and pons. To dissect expression within various neuronal populations, we conducted a comprehensive spatial profiling of Gprc5b across excitatory and inhibitory neuronal types in medial prefrontal cortex, motor cortex, hippocampal regions, hypothalamus, and cerebellum. Overall, we discovered that GABAergic neurons displayed higher Gprc5b expression levels than glutamatergic neurons in most of the analyzed regions with the important exception of the hippocampal dentate gyrus. Overall, the expression analysis of GPRC5B in mouse brain will guide functional studies ultimately positioning GPRC5B in pathophysiological mechanisms and drug discovery.

Development, diagnostic performance, and interobserver agreement of a 18F-flurpiridaz PET automated perfusion quantitation system.

BACKGROUND: Computerized methodologies standardize the myocardial perfusion imaging (MPI) interpretation process. METHODS: To develop an automated relative perfusion quantitation approach for 18F-flurpiridaz, PET MPI studies from all phase III trial participants of 18F-flurpiridaz were divided into 3 groups. Count distributions were obtained in N = 40 normal patients undergoing pharmacological or exercise stress. Then, N = 90 additional studies were selected in a derivation group. Following receiver operating characteristic curve analysis, various standard deviations below the mean normal were used as cutoffs for significant CAD, and interobserver variability determined. Finally, diagnostic performance was compared between blinded visual readers and blinded derivations of automated relative quantitation in the remaining N = 548 validation patients. RESULTS: Both approaches yielded comparable accuracies for the detection of global CAD, reaching 71% and 72% by visual reads, and 72% and 68% by automated relative quantitation, when using CAD ≥ 70% or ≥ 50% stenosis for significance, respectively. Similar results were observed when analyzing individual coronary territories. In both pharmacological and exercise stress, automated relative quantitation demonstrated significantly more interobserver agreement than visual reads. CONCLUSIONS: Our automated method of 18F-flurpiridaz relative perfusion analysis provides a quantitative, objective, and highly reproducible assessment of PET MPI in normal and CAD subjects undergoing either pharmacological or exercise stress.

In vitro profiling of orphan G protein coupled receptor (GPCR) constitutive activity.

BACKGROUND AND PURPOSE: Members of the GPCR family are targeted by a significant fraction of the available FDA-approved drugs. However, the physiological role and pharmacological properties of many GPCRs remain unknown, representing untapped potential in drug design. Of particular interest are ~100 less-studied GPCRs known as orphans because their endogenous ligands are unknown. Intriguingly, disease-causing mutations identified in patients, together with animal studies, have demonstrated that many orphan receptors play crucial physiological roles and, thus, represent attractive drug targets. EXPERIMENTAL APPROACH: The majority of deorphanized GPCRs demonstrate coupling to Gi/o . However, a limited number of techniques allow the detection of intrinsically small constitutive activity associated with Gi/o protein activation, which represents a significant barrier in our ability to study orphan GPCR signalling. Using luciferase reporter assays, we effectively detected constitutive Gs , Gq and G12/13 protein signalling by unliganded receptors and introducing various G protein chimeras, we provide a novel, highly sensitive tool capable of identifying Gi/o coupling in unliganded orphan GPCRs. KEY RESULTS: Using this approach, we measured the constitutive activity of the entire class C GPCR family that includes eight orphan receptors and a subset of 20 prototypical class A GPCR members, including 11 orphans. Excitingly, this approach illuminated the G protein coupling profile of eight orphan GPCRs (GPR22, GPR137b, GPR88, GPR156, GPR158, GPR179, GPRC5D and GPRC6A) previously linked to pathophysiological processes. CONCLUSION AND IMPLICATIONS: We provide a new platform that could be utilized in ongoing studies in orphan receptor signalling and de-orphanization efforts.

First Experience Using 18F-Flubrobenguane PET Imaging in Patients with Suspected Pheochromocytoma or Paraganglioma.

Pheochromocytomas and paragangliomas are a rare tumor entity originating from adrenomedullary chromaffin cells in the adrenal medulla or in sympathetic, paravertebral ganglia outside the medulla. Small lesions are especially difficult to detect by conventional CT or MRI and even by SPECT with the currently available radiotracers (e.g., metaiodobenzylguanidine [MIBG]). The novel PET radiotracer 18F-flubrobenguane could change the diagnostic paradigm in suspected pheochromocytomas and paragangliomas because of its homology with MIBG and the general advantages of PET imaging. The aim of this retrospective analysis was to evaluate 18F-flubrobenguane in pheochromocytomas and paragangliomas and to investigate the biodistribution in patients. Methods: Twenty-three patients with suspected pheochromocytoma or paraganglioma underwent PET/CT or PET/MRI at 63 ± 24 min after injection of 256 ± 33 MBq of 18F-flubrobenguane. The SUVmean and SUVmax of organs were measured with spheric volumes of interest. Threshold-segmented volumes of interest were used to measure the SUVmean or SUVmax of the tumor lesions. One reader evaluated all cross-sectional imaging datasets (CT or MRI) separately, as well as the PET hybrid datasets, and reported the lesion number and size. The diagnostic certainty for a positive lesion was scored on a 3-point scale. Results:18F-flubrobenguane showed a reproducible, stable biodistribution, with the highest SUVmax and SUVmean being in the thyroid gland (30.3 ± 2.2 and 22.5 ± 1.6, respectively), pancreas (12.2 ± 0.8 and 9.5 ± 0.7, respectively), and tumor lesions (16.8 ± 1.7 and 10.1 ± 1.1, respectively) and the lowest SUVmax and SUVmean being in muscle (1.1 ± 0.06 and 0.7 ± 0.04, respectively) and the lung (2.5 ± 0.17 and 1.85 ± 0.13, respectively). In a subgroup analysis, a significantly higher average SUVmean was seen for both pheochromocytoma and paraganglioma than for healthy adrenal glands (11.9 ± 2.0 vs. 9.9 ± 1.5 vs. 3.7 ± 0.2, respectively). In total, 47 lesions were detected. The reader reported more and smaller lesions with higher certainty in PET hybrid imaging than in conventional imaging; however, statistical significance was not reached. Of the 23 (23/47, 49%) lesions smaller than 1 cm, 61% (14/23) were found on hybrid imaging only. Conclusion: Our preliminary data suggest 18F-flubrobenguane PET to be a new, effective staging tool for patients with suspected pheochromocytoma or paraganglioma. Major advantages are the fast acquisition and high spatial resolution of PET imaging and the intense uptake in tumor lesions, facilitating detection. Further studies are warranted to define the role of 18F-flubrobenguane PET, particularly in comparison to standard diagnostic procedures such as MRI or 123I-MIBG SPECT/CT.

Diagnostic Performance of PET Versus SPECT Myocardial Perfusion Imaging in Patients with Smaller Left Ventricles: A Substudy of the 18F-Flurpiridaz Phase III Clinical Trial.

The performance of SPECT myocardial perfusion imaging (MPI) may deteriorate in smaller hearts, primarily because of the lower resolution of conventional Anger cameras. 18F-flurpiridaz is a novel PET MPI agent with superior image and defect resolution. We sought to determine the diagnostic performance of 99mTc-labeled SPECT MPI compared with 18F-flurpiridaz PET MPI according to left ventricle (LV) size. Methods: We conducted a substudy of the phase III clinical trial of flurpiridaz (n = 750) and stratified diagnostic performance according to the median PET LV end-diastolic volume (LVEDV), with smaller LVs defined as having an LVEDV of less than 113 mL (n = 369) and larger LVs defined as having an LVEDV of at least 113 mL (n = 381). Images were interpreted by the majority rule of 3 independent masked readers. The reference standard was quantitative invasive angiography, with at least 50% stenosis in at least 1 coronary artery considered significant. Results: SPECT performance decreased significantly from an area under the curve (AUC) of 0.75 in larger LVs to 0.67 in smaller LVs (P = 0.03), whereas PET performance was similar in larger and smaller LVs (AUC, 0.79 vs. 0.77, P = 0.49). Accordingly, in smaller LVs, PET had a higher AUC (0.77) than the SPECT AUC (0.67) (P < 0.0001), a phenomenon driven by female patients (P < 0.0001). In smaller LVs, there was a degradation of SPECT sensitivity that was highly significant (P < 0.001), whereas there was no significant change in PET sensitivity according to LV size (P = 0.07). Overall, PET had significantly higher sensitivity than SPECT in both smaller LVs (67% vs. 43%, P < 0.001) and larger LVs (76% vs. 61%, P < 0.001). The specificities of PET and SPECT were similar in larger LVs (76% vs. 83%, P = 0.11). Although SPECT specificity improved in smaller compared with larger LVs (90% vs. 83%, P = 0.03), the PET specificity did not change with LV size (76% vs. 76%, P = 0.9). Conclusion: The diagnostic performance of 18F-flurpiridaz PET MPI is not affected by LV size and is superior to SPECT MPI in patients with smaller LVs, highlighting the importance of appropriate test selection in these patients.

Interplay between cell-adhesion molecules governs synaptic wiring of cone photoreceptors.

Establishment of functional synaptic connections in a selective manner is essential for nervous system operation. In mammalian retinas, rod and cone photoreceptors form selective synaptic connections with different classes of bipolar cells (BCs) to propagate light signals. While there has been progress in elucidating rod wiring, molecular mechanisms used by cones to establish functional synapses with BCs have remained unknown. Using an unbiased proteomic strategy in cone-dominant species, we identified the cell-adhesion molecule ELFN2 to be pivotal for the functional wiring of cones with the ON type of BC. It is selectively expressed in cones and transsynaptically recruits the key neurotransmitter receptor mGluR6 in ON-BCs to enable synaptic transmission. Remarkably, ELFN2 in cone terminals functions in synergy with a related adhesion molecule, ELFN1, and their concerted interplay during development specifies selective wiring and transmission of cone signals. These findings identify a synaptic connectivity mechanism of cones and illustrate how interplay between adhesion molecules and postsynaptic transmitter receptors orchestrates functional synaptic specification in a neural circuit.

Phase-III Clinical Trial of Fluorine-18 Flurpiridaz Positron Emission Tomography for Evaluation of Coronary Artery Disease.

BACKGROUND: Fluorine-18 flurpiridaz is a novel positron emission tomography (PET) myocardial perfusion imaging tracer. OBJECTIVES: This study sought to assess the diagnostic efficacy of flurpiridaz PET versus technetium-99m-labeled single photon emission computed tomography SPECT for the detection and evaluation of coronary artery disease (CAD), defined as ≥50% stenosis by quantitative invasive coronary angiography (ICA). Flurpiridaz safety was also evaluated. METHODS: In this phase III prospective multicenter clinical study, 795 patients with known or suspected CAD from 72 clinical sites in the United States, Canada, and Finland were enrolled. A total of 755 patients were evaluable, and the mean age was 62.3 ± 9.5 years, 31% were women, 55% had body mass index ≥30 kg/m2, and 71% had pharmacological stress. Patients underwent 1-day rest-stress (pharmacological or exercise) flurpiridaz PET and 1- or 2-day rest-stress Tc-99m-labeled SPECT and ICA. Images were read by 3 experts blinded to clinical and ICA data. RESULTS: Sensitivity of flurpiridaz PET (for detection of ≥50% stenosis by ICA) was 71.9% (95% confidence interval [CI]: 67.0% to 76.3%), significantly (p < 0.001) higher than SPECT (53.7% [95% CI: 48.5% to 58.8%]), while specificity did not meet the prespecified noninferiority criterion (76.2% [95% CI: 71.8% to 80.1%] vs. 86.6% [95% CI: 83.2% to 89.8%]; p = NS). Receiver-operating characteristic curve analysis demonstrated superior discrimination of CAD by flurpiridaz PET versus SPECT in the overall population, in women, obese patients, and patients undergoing pharmacological stress testing (p < 0.001 for all). Flurpiridaz PET was superior to SPECT for defect size (p < 0.001), image quality (p < 0.001), diagnostic certainty (p < 0.001), and radiation exposure (6.1 ± 0.4 mSv vs. 13.4 ± 3.2 mSv; p < 0.001). Flurpiridaz PET was safe and well tolerated. CONCLUSIONS: Flurpiridaz PET myocardial perfusion imaging shows promise as a new tracer for CAD detection and assessment of women, obese patients, and patients undergoing pharmacological stress testing. A second phase III Food and Drug Administration trial is ongoing. (A Phase 3 Multi-center Study to Assess PET Imaging of Flurpiridaz F 18 Injection in Patients with CAD; NCT01347710).

Orphan G Protein Coupled Receptors in Affective Disorders.

G protein coupled receptors (GPCRs) are the main mediators of signal transduction in the central nervous system. Therefore, it is not surprising that many GPCRs have long been investigated for their role in the development of anxiety and mood disorders, as well as in the mechanism of action of antidepressant therapies. Importantly, the endogenous ligands for a large group of GPCRs have not yet been identified and are therefore known as orphan GPCRs (oGPCRs). Nonetheless, growing evidence from animal studies, together with genome wide association studies (GWAS) and post-mortem transcriptomic analysis in patients, pointed at many oGPCRs as potential pharmacological targets. Among these discoveries, we summarize in this review how emotional behaviors are modulated by the following oGPCRs: ADGRB2 (BAI2), ADGRG1 (GPR56), GPR3, GPR26, GPR37, GPR50, GPR52, GPR61, GPR62, GPR88, GPR135, GPR158, and GPRC5B.

Correction to: PET imaging of sympathetic innervation with [18F]Flubrobenguane vs [11C]mHED in a patient with ischemic cardiomyopathy.

At time of initial publication, the USAN Council had assigned the generic name for LMI1195 as Flurobenguan. However, the Council has since changed and finalized this compound name as Flubrobenguane which is recommended as the generic name to be used in the future.

NF1-cAMP signaling dissociates cell type-specific contributions of striatal medium spiny neurons to reward valuation and motor control.

The striatum plays a fundamental role in motor learning and reward-related behaviors that are synergistically shaped by populations of D1 dopamine receptor (D1R)- and D2 dopamine receptor (D2R)-expressing medium spiny neurons (MSNs). How various neurotransmitter inputs converging on common intracellular pathways are parsed out to regulate distinct behavioral outcomes in a neuron-specific manner is poorly understood. Here, we reveal that distinct contributions of D1R-MSNs and D2R-MSNs towards reward and motor behaviors are delineated by the multifaceted signaling protein neurofibromin 1 (NF1). Using genetic mouse models, we show that NF1 in D1R-MSN modulates opioid reward, whereas loss of NF1 in D2R-MSNs delays motor learning by impeding the formation and consolidation of repetitive motor sequences. We found that motor learning deficits upon NF1 loss were associated with the disruption in dopamine signaling to cAMP in D2R-MSN. Restoration of cAMP levels pharmacologically or chemogenetically rescued the motor learning deficits seen upon NF1 loss in D2R-MSN. Our findings illustrate that multiplex signaling capabilities of MSNs are deployed at the level of intracellular pathways to achieve cell-specific control over behavioral outcomes.

ELFN2 is a postsynaptic cell adhesion molecule with essential roles in controlling group III mGluRs in the brain and neuropsychiatric behavior.

The functional characterization of the GPCR interactome has predominantly focused on intracellular binding partners; however, the recent emergence of transsynaptic GPCR complexes represents an additional dimension to GPCR function that has previously been unaccounted for in drug discovery. Here, we characterize ELFN2 as a novel postsynaptic adhesion molecule with a distinct expression pattern throughout the brain and a selective binding with group III metabotropic glutamate receptors (mGluRs) in trans. Using a transcellular GPCR signaling platform, we report that ELFN2 critically alters group III mGluR secondary messenger signaling by directly altering G protein coupling kinetics and efficacy. Loss of ELFN2 in mice results in the selective downregulation of group III mGluRs and dysregulated glutamatergic synaptic transmission. Elfn2 knockout (Elfn2 KO) mice also feature a range of neuropsychiatric manifestations including seizure susceptibility, hyperactivity, and anxiety/compulsivity, which can be rescued by pharmacological augmentation of group III mGluRs. Thus, we conclude that extracellular transsynaptic scaffolding by ELFN2 in the brain is a cardinal organizational feature of group III mGluRs essential for their signaling properties and brain function.

Beyond the Ligand: Extracellular and Transcellular G Protein-Coupled Receptor Complexes in Physiology and Pharmacology.

G protein-coupled receptors (GPCRs) remain one of the most successful targets of U.S. Food and Drug Administration-approved drugs. GPCR research has predominantly focused on the characterization of the intracellular interactome's contribution to GPCR function and pharmacology. However, emerging evidence uncovers a new dimension in the biology of GPCRs involving their extracellular and transcellular interactions that critically impact GPCR function and pharmacology. The seminal examples include a variety of adhesion GPCRs, such as ADGRLs/latrophilins, ADGRBs/brain angiogenesis inhibitors, ADGRG1/GPR56, ADGRG6/GPR126, ADGRE5/CD97, and ADGRC3/CELSR3. However, recent advances have indicated that class C GPCRs that contain large extracellular domains, including group III metabotropic glutamate receptors (mGluR4, mGluR6, mGluR7, mGluR8), γ-aminobutyric acid receptors, and orphans GPR158 and GPR179, can also participate in this form of transcellular regulation. In this review, we will focus on a variety of identified extracellular and transcellular GPCR-interacting partners, including teneurins, neurexins, integrins, fibronectin leucine-rich transmembranes, contactin-6, neuroligin, laminins, collagens, major prion protein, amyloid precursor protein, complement C1q-likes, stabilin-2, pikachurin, dystroglycan, complement decay-accelerating factor CD55, cluster of differentiation CD36 and CD90, extracellular leucine-rich repeat and fibronectin type III domain containing 1, and leucine-rich repeat, immunoglobulin-like domain and transmembrane domains. We provide an account on the diversity of extracellular and transcellular GPCR complexes and their contribution to key cellular and physiologic processes, including cell migration, axon guidance, cellular and synaptic adhesion, and synaptogenesis. Furthermore, we discuss models and mechanisms by which extracellular GPCR assemblies may regulate communication at cellular junctions. SIGNIFICANCE STATEMENT: G protein-coupled receptors (GPCRs) continue to be the prominent focus of pharmacological intervention for a variety of human pathologies. Although the majority of GPCR research has focused on the intracellular interactome, recent advancements have identified an extracellular dimension of GPCR modulation that alters accepted pharmacological principles of GPCRs. Herein, we describe known endogenous allosteric modulators acting on GPCRs both in cis and in trans.

Genetic behavioral screen identifies an orphan anti-opioid system.

Opioids target the µ-opioid receptor (MOR) to produce unrivaled pain management, but their addictive properties can lead to severe abuse. We developed a whole-animal behavioral platform for unbiased discovery of genes influencing opioid responsiveness. Using forward genetics in Caenorhabditis elegans, we identified a conserved orphan receptor, GPR139, with anti-opioid activity. GPR139 is coexpressed with MOR in opioid-sensitive brain circuits, binds to MOR, and inhibits signaling to heterotrimeric guanine nucleotide-binding proteins (G proteins). Deletion of GPR139 in mice enhanced opioid-induced inhibition of neuronal firing to modulate morphine-induced analgesia, reward, and withdrawal. Thus, GPR139 could be a useful target for increasing opioid safety. These results also demonstrate the potential of C. elegans as a scalable platform for genetic discovery of G protein-coupled receptor signaling principles.

The signaling proteins GPR158 and RGS7 modulate excitability of L2/3 pyramidal neurons and control A-type potassium channel in the prelimbic cortex.

Stress profoundly affects physiological properties of neurons across brain circuits and thereby increases the risk for depression. However, the molecular and cellular mechanisms mediating these effects are poorly understood. In this study, we report that chronic physical restraint stress in mice decreases excitability specifically in layer 2/3 of pyramidal neurons within the prelimbic subarea of the prefrontal cortex (PFC) accompanied by the induction of depressive-like behavioral states. We found that a complex between G protein-coupled receptor (GPCR) 158 (GPR158) and regulator of G protein signaling 7 (RGS7), a regulatory GPCR signaling node recently discovered to be a key modulator of affective behaviors, plays a key role in controlling stress-induced changes in excitability in this neuronal population. Deletion of GPR158 or RGS7 enhanced excitability of layer 2/3 PFC neurons and prevented the impact of stress. Investigation of the underlying molecular mechanisms revealed that the A-type potassium channel Kv4.2 subunit is a molecular target of the GPR158-RGS7 complex. We further report that GPR158 physically associates with Kv4.2 channel and promotes its function by suppressing inhibitory modulation by cAMP-protein kinase A (PKA)-mediated phosphorylation. Taken together, our observations reveal a critical mechanism that adjusts neuronal excitability in L2/3 pyramidal neurons of the PFC and may thereby modulate the effects of stress on depression.

Homeostatic cAMP regulation by the RGS7 complex controls depression-related behaviors.

Affective disorders arise from abnormal responses of the brain to prolonged exposure to challenging environmental stimuli. Recent work identified the orphan receptor GPR158 as a molecular link between chronic stress and depression. Here we reveal a non-canonical mechanism by which GPR158 exerts its effects on stress-induced depression by the complex formation with Regulator of G protein Signaling 7 (RGS7). Chronic stress promotes membrane recruitment of RGS7 via GPR158 in the medial prefrontal cortex (mPFC). The resultant complex suppresses homeostatic regulation of cAMP by inhibitory GPCRs in the region. Accordingly, RGS7 loss in mice induces an antidepressant-like phenotype and resiliency to stress, whereas its restoration within the mPFC is sufficient to rescue this phenotype in a GPR158-dependent way. These findings mechanistically link the unusual orphan receptor-RGS complex to a major stress mediator, the cAMP system and suggest new avenues for pharmacological interventions in affective disorders.