A Global Map of G Protein Signaling Regulation by RGS Proteins.

The control over the extent and timing of G protein signaling is provided by the regulator of G protein signaling (RGS) proteins that deactivate G protein α subunits (Gα). Mammalian genomes encode 20 canonical RGS and 16 Gα genes with key roles in physiology and disease. To understand the principles governing the selectivity of Gα regulation by RGS, we examine the catalytic activity of all canonical human RGS proteins and their selectivity for a complete set of Gα substrates using real-time kinetic measurements in living cells. The data reveal rules governing RGS-Gα recognition, the structural basis of its selectivity, and provide principles for engineering RGS proteins with defined selectivity. The study also explores the evolution of RGS-Gα selectivity through ancestral reconstruction and demonstrates how naturally occurring non-synonymous variants in RGS alter signaling. These results provide a blueprint for decoding signaling selectivity and advance our understanding of molecular recognition principles.

Pharmacogenomics of GPCR Drug Targets.

Natural genetic variation in the human genome is a cause of individual differences in responses to medications and is an underappreciated burden on public health. Although 108 G-protein-coupled receptors (GPCRs) are the targets of 475 (∼34%) Food and Drug Administration (FDA)-approved drugs and account for a global sales volume of over 180 billion US dollars annually, the prevalence of genetic variation among GPCRs targeted by drugs is unknown. By analyzing data from 68,496 individuals, we find that GPCRs targeted by drugs show genetic variation within functional regions such as drug- and effector-binding sites in the human population. We experimentally show that certain variants of µ-opioid and Cholecystokinin-A receptors could lead to altered or adverse drug response. By analyzing UK National Health Service drug prescription and sales data, we suggest that characterizing GPCR variants could increase prescription precision, improving patients' quality of life, and relieve the economic and societal burden due to variable drug responsiveness. VIDEO ABSTRACT.

Fine-tuning GPCR-mediated neuromodulation by biasing signaling through different G protein subunits.

G-protein-coupled receptors (GPCRs) mediate neuromodulation through the activation of heterotrimeric G proteins (Gαßγ). Classical models depict that G protein activation leads to a one-to-one formation of Gα-GTP and Gßγ species. Each of these species propagates signaling by independently acting on effectors, but the mechanisms by which response fidelity is ensured by coordinating Gα and Gßγ responses remain unknown. Here, we reveal a paradigm of G protein regulation whereby the neuronal protein GINIP (Gα inhibitory interacting protein) biases inhibitory GPCR responses to favor Gßγ over Gα signaling. Tight binding of GINIP to Gαi-GTP precludes its association with effectors (adenylyl cyclase) and, simultaneously, with regulator-of-G-protein-signaling (RGS) proteins that accelerate deactivation. As a consequence, Gαi-GTP signaling is dampened, whereas Gßγ signaling is enhanced. We show that this mechanism is essential to prevent the imbalances of neurotransmission that underlie increased seizure susceptibility in mice. Our findings reveal an additional layer of regulation within a quintessential mechanism of signal transduction that sets the tone of neurotransmission.

Mechanisms of Gßγ Release upon GPCR Activation.

Gßγ release is a key event in the transduction of GPCR signals. However, the molecular mechanisms of this process have been unclear. A recent report by Knight et al. provides important clues into the sequence of events that lead to the liberation of Gßγ upon G protein activation by GPCRs.

Members of the KCTD family are major regulators of cAMP signaling.

Cyclic adenosine monophosphate (cAMP) is a pivotal second messenger with an essential role in neuronal function. cAMP synthesis by adenylyl cyclases (AC) is controlled by G protein-coupled receptor (GPCR) signaling systems. However, the network of molecular players involved in the process is incompletely defined. Here, we used CRISPR/Cas9-based screening to identify that members of the potassium channel tetradimerization domain (KCTD) family are major regulators of cAMP signaling. Focusing on striatal neurons, we show that the dominant isoform KCTD5 exerts its effects through an unusual mechanism that modulates the influx of Zn2+ via the Zip14 transporter to exert unique allosteric effects on AC. We further show that KCTD5 controls the amplitude and sensitivity of stimulatory GPCR inputs to cAMP production by Gßγ-mediated AC regulation. Finally, we report that KCTD5 haploinsufficiency in mice leads to motor deficits that can be reversed by chelating Zn2+ Together, our findings uncover KCTD proteins as major regulators of neuronal cAMP signaling via diverse mechanisms.

Genetic modeling of GNAO1 disorder delineates mechanisms of Gαo dysfunction.

GNAO1 encephalopathy is a neurodevelopmental disorder with a spectrum of symptoms that include dystonic movements, seizures and developmental delay. While numerous GNAO1 mutations are associated with this disorder, the functional consequences of pathological variants are not completely understood. Here, we deployed the invertebrate C. elegans as a whole-animal behavioral model to study the functional effects of GNAO1 disorder-associated mutations. We tested several pathological GNAO1 mutations for effects on locomotor behaviors using a combination of CRISPR/Cas9 gene editing and transgenic overexpression in vivo. We report that all three mutations tested (G42R, G203R and R209C) result in strong loss of function defects when evaluated as homozygous CRISPR alleles. In addition, mutations produced dominant negative effects assessed using both heterozygous CRISPR alleles and transgenic overexpression. Experiments in mice confirmed dominant negative effects of GNAO1 G42R, which impaired numerous motor behaviors. Thus, GNAO1 pathological mutations result in conserved functional outcomes across animal models. Our study further establishes the molecular genetic basis of GNAO1 encephalopathy, and develops a CRISPR-based pipeline for functionally evaluating mutations associated with neurodevelopmental disorders.

Severity of GNAO1-Related Disorder Correlates with Changes in G-Protein Function.

OBJECTIVE: GNAO1-related disorders (OMIM #615473 and #617493), caused by variants in the GNAO1 gene, are characterized by developmental delay or intellectual disability, hypotonia, movement disorders, and epilepsy. Neither a genotype-phenotype correlation nor a clear severity score have been established for this disorder. The objective of this prospective and retrospective observational study was to develop a severity score for GNAO1-related disorders, and to delineate the correlation between the underlying molecular mechanisms and clinical severity. METHODS: A total of 16 individuals with GNAO1-related disorders harboring 12 distinct missense variants, including four novel variants (p.K46R, p.T48I, p.R209P, and p.L235P), were examined with repeated clinical assessments, video-electroencephalogram monitoring, and brain magnetic resonance imaging. The molecular pathology of each variant was delineated using a molecular deconvoluting platform. RESULTS: The patients displayed a wide variability in the severity of their symptoms. This heterogeneity was well represented in the GNAO1-related disorders severity score, with a broad range of results. Patients with the same variant had comparable severity scores, indicating that differences in disease profiles are not due to interpatient variability, but rather, to unique disease mechanisms. Moreover, we found a significant correlation between clinical severity scores and molecular mechanisms. INTERPRETATION: The clinical score proposed here provides further insight into the correlation between pathophysiology and phenotypic severity in GNAO1-related disorders. We found that each variant has a unique profile of clinical phenotypes and pathological molecular mechanisms. These findings will contribute to better understanding GNAO1-related disorders. Additionally, the severity score will facilitate standardization of patients categorization and assessment of response to therapies in development. ANN NEUROL 2023;94:987-1004.

Adhesion GPCR Latrophilin 3 regulates synaptic function of cone photoreceptors in a trans-synaptic manner.

Cone photoreceptors mediate daylight vision in vertebrates. Changes in neurotransmitter release at cone synapses encode visual information and is subject to precise control by negative feedback from enigmatic horizontal cells. However, the mechanisms that orchestrate this modulation are poorly understood due to a virtually unknown landscape of molecular players. Here, we report a molecular player operating selectively at cone synapses that modulates effects of horizontal cells on synaptic release. Using an unbiased proteomic screen, we identified an adhesion GPCR Latrophilin3 (LPHN3) in horizontal cell dendrites that engages in transsynaptic control of cones. We detected and characterized a prominent splice isoform of LPHN3 that excludes a element with inhibitory influence on transsynaptic interactions. A gain-of-function mouse model specifically routing LPHN3 splicing to this isoform but not knockout of LPHN3 diminished CaV1.4 calcium channel activity profoundly disrupted synaptic release by cones and resulted in synaptic transmission deficits. These findings offer molecular insight into horizontal cell modulation on cone synaptic function and more broadly demonstrate the importance of alternative splicing in adhesion GPCRs for their physiological function.

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.

GPCR-dependent biasing of GIRK channel signaling dynamics by RGS6 in mouse sinoatrial nodal cells.

How G protein-coupled receptors (GPCRs) evoke specific biological outcomes while utilizing a limited array of G proteins and effectors is poorly understood, particularly in native cell systems. Here, we examined signaling evoked by muscarinic (M2R) and adenosine (A1R) receptor activation in the mouse sinoatrial node (SAN), the cardiac pacemaker. M2R and A1R activate a shared pool of cardiac G protein-gated inwardly rectifying K+ (GIRK) channels in SAN cells from adult mice, but A1R-GIRK responses are smaller and slower than M2R-GIRK responses. Recordings from mice lacking Regulator of G protein Signaling 6 (RGS6) revealed that RGS6 exerts a GPCR-dependent influence on GIRK-dependent signaling in SAN cells, suppressing M2R-GIRK coupling efficiency and kinetics and A1R-GIRK signaling amplitude. Fast kinetic bioluminescence resonance energy transfer assays in transfected HEK cells showed that RGS6 prefers Gαo over Gαi as a substrate for its catalytic activity and that M2R signals preferentially via Gαo, while A1R does not discriminate between inhibitory G protein isoforms. The impact of atrial/SAN-selective ablation of Gαo or Gαi2 was consistent with these findings. Gαi2 ablation had minimal impact on M2R-GIRK and A1R-GIRK signaling in SAN cells. In contrast, Gαo ablation decreased the amplitude and slowed the kinetics of M2R-GIRK responses, while enhancing the sensitivity and prolonging the deactivation rate of A1R-GIRK signaling. Collectively, our data show that differences in GPCR-G protein coupling preferences, and the Gαo substrate preference of RGS6, shape A1R- and M2R-GIRK signaling dynamics in mouse SAN cells.

Effects of Small Molecule Ligands on ACKR3 Receptors.

Chemokines such as stromal derived factor 1 and their G protein coupled receptors are well-known regulators of the development and functions of numerous tissues. C-X-C motif chemokine ligand 12 (CXCL12) has two receptors: C-X-C chemokine motif receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3). ACKR3 has been described as an atypical "biased" receptor because it does not appear to signal through G proteins and, instead, signals solely through the ß-arrestin pathway. In support of this conclusion, we have shown that ACKR3 is unable to signal through any of the known mammalian G α isoforms and have generated a comprehensive map of the G α activation by CXCL12/CXCR4. We also synthesized a series of small molecule ligands which acted as selective agonists for ACKR3 as assessed by their ability to recruit ß-arrestin to the receptor. Using select point mutations, we studied the molecular characteristics that determine the ability of small molecules to activate ACKR3 receptors, revealing a key role for the deeper binding pocket composed of residues in the transmembrane domains of ACKR3. The development of more selective ACKR3 ligands should allow us to better appreciate the unique roles of ACKR3 in the CXCL12/CXCR4/ACKR3-signaling axis and better understand the structural determinants for ACKR3 activation. SIGNIFICANCE STATEMENT: We are interested in the signaling produced by the G protein coupled receptor atypical chemokine receptor 3 (ACKR3), which signals atypically. In this study, novel selective ligands for ACKR3 were discovered and the site of interactions between these small molecules and ACKR3 was defined. This work will help to better understand the unique signaling roles of ACKR3.

Middle-Down Mass Spectrometry Reveals Activity-Modifying Phosphorylation Barcode in a Class C G Protein-Coupled Receptor.

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in humans. They mediate nearly all aspects of human physiology and thus are of high therapeutic interest. GPCR signaling is regulated in space and time by receptor phosphorylation. It is believed that different phosphorylation states are possible for a single receptor, and each encodes for unique signaling outcomes. Methods to determine the phosphorylation status of GPCRs are critical for understanding receptor physiology and signaling properties of GPCR ligands and therapeutics. However, common proteomic techniques have provided limited quantitative information regarding total receptor phosphorylation stoichiometry, relative abundances of isomeric modification states, and temporal dynamics of these parameters. Here, we report a novel middle-down proteomic strategy and parallel reaction monitoring (PRM) to quantify the phosphorylation states of the C-terminal tail of metabotropic glutamate receptor 2 (mGluR2). By this approach, we found that mGluR2 is subject to both basal and agonist-induced phosphorylation at up to four simultaneous sites with varying probability. Using a PRM tandem mass spectrometry methodology, we localized the positions and quantified the relative abundance of phosphorylations following treatment with an agonist. Our analysis showed that phosphorylation within specific regions of the C-terminal tail of mGluR2 is sensitive to receptor activation, and subsequent site-directed mutagenesis of these sites identified key regions which tune receptor sensitivity. This study demonstrates that middle-down purification followed by label-free quantification is a powerful, quantitative, and accessible tool for characterizing phosphorylation states of GPCRs and other challenging proteins.

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.

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.

The orphan receptor GPR139 signals via Gq/11 to oppose opioid effects.

The interplay between G protein-coupled receptors (GPCRs) is critical for controlling neuronal activity that shapes neuromodulatory outcomes. Recent evidence indicates that the orphan receptor GPR139 influences opioid modulation of key brain circuits by opposing the actions of the µ-opioid receptor (MOR). However, the function of GPR139 and its signaling mechanisms are poorly understood. In this study, we report that GPR139 activates multiple heterotrimeric G proteins, including members of the Gq/11 and Gi/o families. Using a panel of reporter assays in reconstituted HEK293T/17 cells, we found that GPR139 functions via the Gq/11 pathway and thereby distinctly regulates cellular effector systems, including stimulation of cAMP production and inhibition of G protein inward rectifying potassium (GIRK) channels. Electrophysiological recordings from medial habenular neurons revealed that GPR139 signaling via Gq/11 is necessary and sufficient for counteracting MOR-mediated inhibition of neuronal firing. These results uncover a mechanistic interplay between GPCRs involved in controlling opioidergic neuromodulation in the brain.

Striatal Dopamine Induced ERK Phosphorylation Is Altered in Mouse Models of Monogenic Dystonia.

BACKGROUND: Similar to some monogenic forms of dystonia, levodopa-induced dyskinesia is a hyperkinetic movement disorder with abnormal nigrostriatal dopaminergic neurotransmission. Molecularly, it is characterized by hyper-induction of phosphorylation of extracellular signal-related kinase in response to dopamine in medium spiny neurons of the direct pathway. OBJECTIVES: The objective of this study was to determine if mouse models of monogenic dystonia exhibit molecular features of levodopa-induced dyskinesia. METHODS: Western blotting and quantitative immunofluorescence was used to assay baseline and/or dopamine-induced levels of the phosphorylated kinase in the striatum in mouse models of DYT1, DYT6, and DYT25 expressing a reporter in dopamine D1 receptor-expressing projection neurons. Cyclic adenosine monophosphate (cAMP) immunoassay and adenylyl cyclase activity assays were also performed. RESULTS: In DYT1 and DYT6 models, blocking dopamine reuptake with cocaine leads to enhanced extracellular signal-related kinase phosphorylation in dorsomedial striatal medium spiny neurons in the direct pathway, which is abolished by pretreatment with the N-methyl-d-aspartate antagonist MK-801. Phosphorylation is decreased in a model of DYT25. Levels of basal and stimulated cAMP and adenylyl cyclase activity were normal in the DYT1 and DYT6 mice and decreased in the DYT25 mice. Oxotremorine induced increased abnormal movements in the DYT1 knock-in mice. CONCLUSIONS: The increased dopamine induction of extracellular signal-related kinase phosphorylation in 2 genetic types of dystonia, similar to what occurs in levodopa-induced dyskinesia, and its decrease in a third, suggests that abnormal signal transduction in response to dopamine in the postsynaptic nigrostriatal pathway might be a point of convergence for dystonia and other hyperkinetic movement disorders, potentially offering common therapeutic targets. © 2021 International Parkinson and Movement Disorder Society.

Clarin-1 expression in adult mouse and human retina highlights a role of Müller glia in Usher syndrome.

Usher syndrome type 3 (USH3) is an autosomal recessively inherited disorder caused by mutations in the gene clarin-1 (CLRN1), leading to combined progressive hearing loss and retinal degeneration. The cellular distribution of CLRN1 in the retina remains uncertain, either because its expression levels are low or because its epitopes are masked. Indeed, in the adult mouse retina, Clrn1 mRNA is developmentally downregulated, detectable only by RT-PCR. In this study we used the highly sensitive RNAscope in situ hybridization assay and single-cell RNA-sequencing techniques to investigate the distribution of Clrn1 and CLRN1 in mouse and human retina, respectively. We found that Clrn1 transcripts in mouse tissue are localized to the inner retina during postnatal development and in adult stages. The pattern of Clrn1 mRNA cellular expression is similar in both mouse and human adult retina, with CLRN1 transcripts being localized in Müller glia, and not photoreceptors. We generated a novel knock-in mouse with a hemagglutinin (HA) epitope-tagged CLRN1 and showed that CLRN1 is expressed continuously at the protein level in the retina. Following enzymatic deglycosylation and immunoblotting analysis, we detected a single CLRN1-specific protein band in homogenates of mouse and human retina, consistent in size with the main CLRN1 isoform. Taken together, our results implicate Müller glia in USH3 pathology, placing this cell type to the center of future mechanistic and therapeutic studies to prevent vision loss in this disease. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

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.