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.

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.

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.

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.

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.

Synaptic adhesion protein ELFN1 is a selective allosteric modulator of group III metabotropic glutamate receptors in trans.

Functional characterization of the GPCR interactome has been focused predominantly on intracellular interactions, 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 of transsynaptic interactions on properties and pharmacology of these receptors are unknown. In the current study, we explore the effects of ELFN1 on mGluR signaling and pharmacology. First, we established the binding specificity of ELFN1 and found it to be recruited selectively to all group III mGluRs (mGluR4, mGluR6, mGluR7, and mGluR8), but not other mGluR species. Using site-directed mutagenesis we mapped binding determinants of this interaction to two distinct sites on the ELFN1 ectodomain. To evaluate functional aspects of the interaction, we developed a transcellular signaling assay in reconstituted HEK293 cells which monitors changes in mGluR activity in one cell following its exposure to separate ELFN1-containing cells. Using this platform, we found that ELFN1 acts as an allosteric modulator of class III mGluR activity in suppressing cAMP accumulation: altering both agonist-induced and constitutive receptor activity. Using bioluminescence resonance energy transfer-based real-time kinetic assays, we established that ELFN1 alters the ability of mGluRs to activate G proteins. Our findings demonstrate that core properties of class III mGluRs can be altered via extracellular interactions with ELFN1 which serves as a transsynaptic allosteric modulator for these receptors. Furthermore, our unique assay platform opens avenues for exploring transcellular/transsynaptic pharmacology of other GPCR transcomplexes.

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.

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.

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.

Inhibitory Signaling to Ion Channels in Hippocampal Neurons Is Differentially Regulated by Alternative Macromolecular Complexes of RGS7.

The neuromodulatory effects of GABA on pyramidal neurons are mediated by GABAB receptors (GABABRs) that signal via a conserved G-protein-coupled pathway. Two prominent effectors regulated by GABABRs include G-protein inwardly rectifying K+ (GIRK) and P/Q/N type voltage-gated Ca2+ (CaV2) ion channels that control excitability and synaptic output of these neurons, respectively. Regulator of G-protein signaling 7 (RGS7) has been shown to control GABAB effects, yet the specificity of its impacts on effector channels and underlying molecular mechanisms is poorly understood. In this study, we show that hippocampal RGS7 forms two distinct complexes with alternative subunit configuration bound to either membrane protein R7BP (RGS7 binding protein) or orphan receptor GPR158. Quantitative biochemical experiments show that both complexes account for targeting nearly the entire pool of RGS7 to the plasma membrane. We analyzed the effect of genetic elimination in mice of both sexes and overexpression of various components of RGS7 complex by patch-clamp electrophysiology in cultured neurons and brain slices. We report that RGS7 prominently regulates GABABR signaling to CaV2, in addition to its known involvement in modulating GIRK. Strikingly, only complexes containing R7BP, but not GPR158, accelerated the kinetics of both GIRK and CaV2 modulation by GABABRs. In contrast, GPR158 overexpression exerted the opposite effect and inhibited RGS7-assisted temporal modulation of GIRK and CaV2 by GABA. Collectively, our data reveal mechanisms by which distinctly composed macromolecular complexes modulate the activity of key ion channels that mediate the inhibitory effects of GABA on hippocampal CA1 pyramidal neurons.SIGNIFICANCE STATEMENT This study identifies the contributions of distinct macromolecular complexes containing a major G-protein regulator to controlling key ion channel function in hippocampal neurons with implications for understanding molecular mechanisms underlying synaptic plasticity, learning, and memory.

Intermolecular Interaction between Anchoring Subunits Specify Subcellular Targeting and Function of RGS Proteins in Retina ON-Bipolar Neurons.

In vertebrate retina, light responses generated by the rod photoreceptors are transmitted to the second-order neurons, the ON-bipolar cells (ON-BC), and this communication is indispensible for vision in dim light. In ON-BCs, synaptic transmission is initiated by the metabotropic glutamate receptor, mGluR6, that signals via the G-protein Go to control opening of the effector ion channel, TRPM1. A key role in this process belongs to the GTPase Activating Protein (GAP) complex that catalyzes Go inactivation upon light-induced suppression of glutamate release in rod photoreceptors, thereby driving ON-BC depolarization to changes in synaptic input. The GAP complex has a striking molecular complexity. It contains two Regulator of G-protein Signaling (RGS) proteins RGS7 and RGS11 that directly act on Go and two adaptor subunits: RGS Anchor Protein (R9AP) and the orphan receptor, GPR179. Here we examined the organizational principles of the GAP complex in ON-BCs. Biochemical experiments revealed that RGS7 binds to a conserved site in GPR179 and that RGS11 in vivo forms a complex only with R9AP. R9AP and GPR179 are further integrated via direct protein-protein interactions involving their cytoplasmic domains. Elimination of GPR179 prevents postsynaptic accumulation of R9AP. Furthermore, concurrent knock-out of both R9AP and RGS7 does not reconfigure the GAP complex and completely abolishes synaptic transmission, resulting in a novel mouse model of night blindness. Based on these results, we propose a model of hierarchical assembly and function of the GAP complex that supports ON-BCs visual signaling.

Regulator of G Protein Signaling 7 (RGS7) Can Exist in a Homo-oligomeric Form That Is Regulated by Gαo and R7-binding Protein.

RGS (regulator of G protein signaling) proteins of the R7 subfamily (RGS6, -7, -9, and -11) are highly expressed in neurons where they regulate many physiological processes. R7 RGS proteins contain several distinct domains and form obligatory dimers with the atypical Gß subunit, Gß5 They also interact with other proteins such as R7-binding protein, R9-anchoring protein, and the orphan receptors GPR158 and GPR179. These interactions facilitate plasma membrane targeting and stability of R7 proteins and modulate their activity. Here, we investigated RGS7 complexes using in situ chemical cross-linking. We found that in mouse brain and transfected cells cross-linking causes formation of distinct RGS7 complexes. One of the products had the apparent molecular mass of ∼150 kDa on SDS-PAGE and did not contain Gß5 Mass spectrometry analysis showed no other proteins to be present within the 150-kDa complex in the amount close to stoichiometric with RGS7. This finding suggested that RGS7 could form a homo-oligomer. Indeed, co-immunoprecipitation of differentially tagged RGS7 constructs, with or without chemical cross-linking, demonstrated RGS7 self-association. RGS7-RGS7 interaction required the DEP domain but not the RGS and DHEX domains or the Gß5 subunit. Using transfected cells and knock-out mice, we demonstrated that R7-binding protein had a strong inhibitory effect on homo-oligomerization of RGS7. In contrast, our data indicated that GPR158 could bind to the RGS7 homo-oligomer without causing its dissociation. Co-expression of constitutively active Gαo prevented the RGS7-RGS7 interaction. These results reveal the existence of RGS protein homo-oligomers and show regulation of their assembly by R7 RGS-binding partners.

Orphan Receptor GPR158 Is an Allosteric Modulator of RGS7 Catalytic Activity with an Essential Role in Dictating Its Expression and Localization in the Brain.

Regulators of G protein signaling control the duration and extent of signaling via G protein-coupled receptor (GPCR) pathways by accelerating the GTP hydrolysis on G protein α subunits thereby promoting termination of GPCR signaling. A member of this family, RGS7, plays a critical role in the nervous system where it regulates multiple neurotransmitter GPCRs that mediate vision, memory, and the action of addictive drugs. Previous studies have established that in vivo RGS7 forms mutually exclusive complexes with the membrane protein RGS7-binding protein or the orphan receptor GPR158. In this study, we examine the impact of GPR158 on RGS7 in the brain. We report that knock-out of GPR158 in mice results in marked post-transcriptional destabilization of RGS7 and substantial loss of its association with membranes in several brain regions. We further identified the RGS7-binding site in the C terminus of GPR158 and found that it shares significant homology with the RGS7-binding protein. The proximal portion of the GPR158 C terminus additionally contained a conserved sequence that was capable of enhancing RGS7 GTPase-activating protein activity in solution by an allosteric mechanism acting in conjunction with the regulators of the G protein signaling-binding domain. The distal portion of the GPR158 C terminus contained several phosphodiesterase E γ-like motifs and selectively recruited G proteins in their activated state. The results of this study establish GPR158 as an essential regulator of RGS7 in the native nervous system with a critical role in controlling its expression, membrane localization, and catalytic activity.

Modulation of dendritic AMPA receptor mRNA trafficking by RNA splicing and editing.

RNA trafficking to dendrites and local translation are crucial processes for superior neuronal functions. To date, several α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) mRNAs have been detected in dendrites and are subject to local protein synthesis. Here, we report the presence of all AMPAR GluA1-4 mRNAs in hippocampal and cortical rat synaptic spines by synaptoneurosomes analysis. In particular, we showed that dendritic AMPAR mRNAs are present in the Flip versions in the cortex and hippocampus. To further confirm these data, we demonstrate, using in situ hybridization, the dendritic localization of the GluA2 Flip isoform in vitro and in vivo, whereas the Flop variant is restricted mainly to the soma. In addition, we report that dendritic AMPA mRNAs are edited at low levels at their R/G sites; this result was also supported with transfection experiments using chimeric GluA2 DNA vectors, showing that transcripts carrying an unedited nucleotide at the R/G site, in combination with the Flip exon, are more efficiently targeted to dendrites when compared with the edited-Flip versions. Our data show that post-transcriptional regulations such as RNA splicing, editing and trafficking might be mutually coordinated and that the localization of different AMPAR isoforms in dendrites might play a functional role in the regulation of neuronal transmission.

Gz Enhanced Signal Transduction assaY (GZESTY) for GPCR deorphanization.

G protein-coupled receptors (GPCRs) are key pharmacological targets, yet many remain underutilized due to unknown activation mechanisms and ligands. Orphan GPCRs, lacking identified natural ligands, are a high priority for research, as identifying their ligands will aid in understanding their functions and potential as drug targets. Most GPCRs, including orphans, couple to Gi/o/z family members, however current assays to detect their activation are limited, hindering ligand identification efforts. We introduce GZESTY, a highly sensitive, cell-based assay developed in an easily deliverable format designed to study the pharmacology of Gi/o/z-coupled GPCRs and assist in deorphanization. We optimized assay conditions and developed an all-in-one vector employing novel cloning methods to ensure the correct expression ratio of GZESTY components. GZESTY successfully assessed activation of a library of ligand-activated GPCRs, detecting both full and partial agonism, as well as responses from endogenous GPCRs. Notably, with GZESTY we established the presence of endogenous ligands for GPR176 and GPR37 in brain extracts, validating its use in deorphanization efforts. This assay enhances the ability to find ligands for orphan GPCRs, expanding the toolkit for GPCR pharmacologists.

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.

Lixivaptan safely and effectively corrects serum sodium concentrations in hospitalized patients with euvolemic hyponatremia.

Hyponatremia is a common electrolyte disorder associated with increased morbidity and mortality, particularly in the elderly. Lixivaptan, a new selective vasopressin V2-receptor antagonist, safely corrected serum sodium concentrations in phase II studies of patients with euvolemic hyponatremia. Here our multinational, double-blind, placebo-controlled, phase III study assessed the effect of lixivaptan on serum sodium concentrations in 106 initially hospitalized patients with euvolemic hyponatremia (serum sodium less than 130 mmol/l). Of them, 52 were randomized to receive placebo and 54 received 50 mg lixivaptan once daily and were then titrated to receive 25-100 mg once daily depending on serum sodium concentration. Fluid restriction was at the investigator's discretion. Initial titration occurred in a monitored inpatient setting; patients were then treated as outpatients for a total of 30 days. The primary end point was the change in serum sodium concentration from baseline to day 7. Lixivaptan significantly increased the serum sodium concentration from baseline to day 7 (the primary end point) by 6.7 mmol/l compared with placebo (4.5 mmol/l; P=0.034). Importantly, the serum sodium concentration was normalized safely and more rapidly in patients receiving lixivaptan than placebo (P=0.004) and was well tolerated. After drug discontinuation, serum sodium concentrations decreased to near-baseline levels within 7 days. Thus, lixivaptan safely and effectively corrects serum sodium concentrations in patients with euvolemic hyponatremia.