Endogenous G Protein-coupled Receptors Research Articles

Direct detection of endogenous Gαi activity in cells with a sensitive conformational biosensor.

Activation of heterotrimeric G-proteins (Gαβγ) by G-protein-coupled receptors (GPCRs) is not only a mechanism broadly used by eukaryotes to transduce signals across the plasma membrane, but also the target for a large fraction of clinical drugs. However, approaches typically used to assess this signaling mechanism by directly measuring G-protein activity, like optical biosensors, suffer from limitations. On one hand, many of these biosensors require expression of exogenous GPCRs and/or G-proteins, compromising readout fidelity. On the other hand, biosensors that measure endogenous signaling may still interfere with the signaling process under investigation or suffer from having a small dynamic range of detection, hindering broad applicability. Here, we developed an optical biosensor that detects the endogenous G-protein active species Gαi-GTP upon stimulation of endogenous GPCRs more robustly than current state-of-the-art sensors for the same purpose. Its design is based on the principle of bystander Bioluminescence Resonance Energy Transfer (BRET) and leverages the Gαi-binding protein named GINIP as a high affinity and specific detector module of the GTP-bound conformation of Gαi. We optimized this design to prevent interference with Gi-dependent signaling (cAMP inhibition) and to enable implementation in different experimental systems with endogenous GPCRs, including neurotransmitter receptors in primary astroglial cells or opioid receptors in cell lines, which revealed opioid neuropeptide-mediated activation profiles different from those observed with other biosensors involving exogenous GPCRs and G-proteins. Overall, we introduce a biosensor that directly and sensitively detects endogenous activation of G-proteins by GPCRs across different experimental settings without interfering with the subsequent propagation of signaling.

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.

Single-chain fluorescent integrators for mapping G-protein-coupled receptor agonists.

G protein-coupled receptors (GPCRs) transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors that are highly modular and could potentially be used to determine GPCR agonist localization across the brain. We previously engineered integrator sensors for the mu- and kappa-opioid receptor agonists called M- and K-Single-chain Protein-based Opioid Transmission Indicator Tool (SPOTIT), respectively. Here, we engineered red versions of the SPOTIT sensors for multiplexed imaging of GPCR agonists. We also modified SPOTIT to create an integrator sensor design platform called SPOTIT for all GPCRs (SPOTall). We used the SPOTall platform to engineer sensors for the beta 2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. Finally, we demonstrated the application of M-SPOTIT and B2AR-SPOTall in detecting exogenously administered morphine, isoproterenol, and epinephrine in the mouse brain via locally injected viruses. The SPOTIT and SPOTall sensor design platform has the potential for unbiased agonist detection of many synthetic and endogenous neuromodulators across the brain.

Ubiquitin-driven G protein-coupled receptor inflammatory signaling at the endosome.

G protein-coupled receptors (GPCRs) are ubiquitously expressed cell surface receptors that mediate numerous physiological responses and are highly druggable. Upon activation, GPCRs rapidly couple to heterotrimeric G proteins and are then phosphorylated and internalized from the cell surface. Recent studies indicate that GPCRs not only localize at the plasma membrane but also exist in intracellular compartments where they are competent to signal. Intracellular signaling by GPCRs is best described to occur at endosomes. Several studies have elegantly documented endosomal GPCR-G protein and GPCR-β-arrestin signaling. Besides phosphorylation, GPCRs are also posttranslationally modified with ubiquitin. GPCR ubiquitination has been studied mainly in the context of receptor endosomal-lysosomal trafficking. However, new studies indicate that ubiquitination of endogenous GPCRs expressed in endothelial cells initiates the assembly of an intracellular p38 mitogen-activated kinase signaling complex that promotes inflammatory responses from endosomes. In this mini-review, we discuss emerging discoveries that provide critical insights into the function of ubiquitination in regulating GPCR inflammatory signaling at endosomes.

2023 Julius Axelrod Symposium: Plant-Derived Molecules Acting on G Protein-Coupled Receptors.

Plant extracts have played a significant role in traditional medicine for centuries, contributing to improved health and the treatment of various human illnesses. G protein-coupled receptors (GPCRs) are crucial in numerous physiologic functions, and there is growing evidence suggesting their involvement in the therapeutic effects of many plant extracts. In recent years, scientists have identified an expanding number of isolated molecules responsible for the biologic activity of these extracts, with many believed to act on GPCRs. This article critically reviews the evidence supporting the modulation of GPCR function by these plant-derived molecules through direct binding. Structural information is now available for some of these molecules, allowing for a comparison of their binding mode with that of endogenous GPCR ligands. The final section explores future trends and challenges, focusing on the identification of new plant-derived molecules with both orthosteric and allosteric binding modes, as well as innovative strategies for designing GPCR ligands inspired by these plant-derived compounds. In conclusion, plant-derived molecules are anticipated to play an increasingly vital role as therapeutic drugs and serve as templates for drug design. SIGNIFICANCE STATEMENT: This minireview summarizes the most pertinent publications on isolated plant-derived molecules interacting with G protein-coupled receptors (GPCRs) and comments on available structural information on GPCR/plant-derived ligand pairs. Future challenges and trends for the isolation and characterization of plant-derived molecules and drug design are discussed.

Specific pharmacological and Gi/o protein responses of some native GPCRs in neurons

G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins and are important drug targets. The discovery of drugs targeting these receptors and their G protein signaling properties are based on assays mainly performed with modified receptors expressed in heterologous cells. However, GPCR responses may differ in their native environment. Here, by using highly sensitive Gi/o sensors, we reveal specific properties of Gi/o protein-mediated responses triggered by GABAB, α2 adrenergic and cannabinoid CB1 receptors in primary neurons, different from those in heterologous cells. These include different profiles in the Gi/o protein subtypes-mediated responses, and differences in the potencies of some ligands even at similar receptor expression levels. Altogether, our results show the importance of using biosensors compatible with primary cells for evaluating the activities of endogenous GPCRs in their native environment.

Direct interrogation of context-dependent GPCR activity with a universal biosensor platform

G protein-coupled receptors (GPCRs) are the largest family of druggable proteins encoded in the human genome, but progress in understanding and targeting them is hindered by the lack of tools to reliably measure their nuanced behavior in physiologically relevant contexts. Here, we developed a collection of compact ONE vector G-protein Optical (ONE-GO) biosensor constructs as a scalable platform that can be conveniently deployed to measure G-protein activation by virtually any GPCR with high fidelity even when expressed endogenously in primary cells. By characterizing dozens of GPCRs across many cell types like primary cardiovascular cells or neurons, we revealed insights into the molecular basis for G-protein coupling selectivity of GPCRs, pharmacogenomic profiles of anti-psychotics on naturally occurring GPCR variants, and G-protein subtype signaling bias by endogenous GPCRs depending on cell type or upon inducing disease-like states. In summary, this open-source platform makes the direct interrogation of context-dependent GPCR activity broadly accessible.

Highly multiplexed bioactivity screening reveals human and microbiota metabolome-GPCRome interactions

The human body contains thousands of metabolites derived from mammalian cells, the microbiota, food, and medical drugs. Many bioactive metabolites act through the engagement of G-protein-coupled receptors (GPCRs); however, technological limitations constrain current explorations of metabolite-GPCR interactions. Here, we developed a highly multiplexed screening technology called PRESTO-Salsa that enables simultaneous assessment of nearly all conventional GPCRs (>300 receptors) in a single well of a 96-well plate. Using PRESTO-Salsa, we screened 1,041 human-associated metabolites against the GPCRome and uncovered previously unreported endogenous, exogenous, and microbial GPCR agonists. Next, we leveraged PRESTO-Salsa to generate an atlas of microbiome-GPCR interactions across 435 human microbiome strains from multiple body sites, revealing conserved patterns of cross-tissue GPCR engagement and activation of CD97/ADGRE5 by the Porphyromonas gingivalis protease gingipain K. These studies thus establish a highly multiplexed bioactivity screening technology and expose a diverse landscape of human, diet, drug, and microbiota metabolome-GPCRome interactions.

Detecting GPCR Signals With Optical Biosensors of Gα-GTP in Cell Lines and Primary Cell Cultures.

G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors and mediate a wide variety of physiological processes. GPCRs respond to a plethora of extracellular ligands and initiate signaling pathways inside cells via heterotrimeric G proteins (Gαβγ). Because of the critical role GPCRs play in regulating biological processes and as pharmacological targets, the availability of tools to measure their signaling activity are of high interest. Live-cell biosensors that detect the activity of G proteins in response to GPCR stimulation have emerged as a powerful approach to investigate GPCR/G protein signaling. Here, we detail methods to monitor G protein activity through direct measurement of GTP-bound Gα subunits using optical biosensors based on bioluminescence resonance energy transfer (BRET). More specifically, this article describes the use of two types of complementary biosensors. The first protocol explains how to use a multicomponent BRET biosensor that relies on expression of exogenous G proteins in cell lines. This protocol yields robust responses that are compatible with endpoint measurements of dose-dependent ligand effects or with kinetic measurements of subsecond resolution. The second protocol describes the implementation of unimolecular biosensors that detect the activation of endogenous G proteins in cell lines expressing exogenous GPCRs or in primary cells upon stimulation of endogenous GPCRs. Overall, using the biosensors as described in this article will help users characterize the mechanisms of action of many pharmacological agents and natural ligands that modulate GPCR and G protein signaling with high precision. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Using bimolecular BRET biosensors to monitor Gα-GTP formation of tagged Gα in live cells Alternate Protocol 1: Measuring GPCR dose-dependent Gα-GTP responses in endpoint format Basic Protocol 2: Using unimolecular BRET biosensors to study endogenous G protein activity Alternate Protocol 2: Using unimolecular BRET biosensors to study endogenous G protein activity in mouse cortical neurons.

In situ visualization of opioid and cannabinoid drug effects using phosphosite-specific GPCR antibodies

G protein-coupled receptors (GPCRs) are important signal transducers that are phosphorylated upon activation at intracellular serine and threonine residues. Although antibodies that specifically recognize the phosphorylation state of GPCRs have been available for many years, efficient immunolocalization of phosphorylated receptors in their tissues of origin has not been possible. Here, we show that phosphorylation of receptors is highly unstable during routine immunohistochemical procedures, requiring the use of appropriate phosphatase inhibitors particular during tissue perfusion, post-fixation, and cryoprotection but not during immunostaining of tissue sections. We provide proof of concept using phosphorylation state-specific μ-opioid receptor (MOP) and cannabinoid receptor 1 (CB1) antibodies. Indeed, three of four well-characterized phosphosite-specific MOP antibodies, including pS375-MOP, pT376-MOP, and pT379-MOP, showed robust neuronal immunostaining in brain and spinal cord sections of opioid-treated mice only after inclusion of phosphatase inhibitors. We then extended this approach to the CB1 receptor and demonstrated that one of three newly-generated phosphosite-specific CB1 antibodies, namely pS425-CB1, showed striking staining of fibers and varicosities in brain slices from cannabinoid-treated mice. Although subsequent experiments showed that phospho-CB1 immunostaining was less sensitive to phosphatases, we conclude that the use of phosphatase inhibitors should always be considered in the development of immunohistochemical procedures for new phosphosite-specific GPCR antibodies. In summary, we anticipate that this improved protocol will facilitate the widespread use of phosphorylation state-specific antibodies to monitor the activation of endogenous GPCRs under physiological and pharmacological conditions. Our approach may also prove useful to confirm target engagement of GPCR drug candidates in native tissues.

Multiple Subthreshold GPCR Signals Combined by the G-Proteins Gαq and Gαs Activate the Caenorhabditis elegans Egg-Laying Muscles.

Individual neurons or muscle cells express many G-protein-coupled receptors (GPCRs) for neurotransmitters and neuropeptides, yet it remains unclear how cells integrate multiple GPCR signals that all must activate the same few G-proteins. We analyzed this issue in the Caenorhabditis elegans egg-laying system, where multiple GPCRs on muscle cells promote contraction and egg laying. We genetically manipulated individual GPCRs and G-proteins specifically in these muscle cells within intact animals and then measured egg laying and muscle calcium activity. Two serotonin GPCRs on the muscle cells, Gαq-coupled SER-1 and Gαs-coupled SER-7, together promote egg laying in response to serotonin. We found that signals produced by either SER-1/Gαq or SER-7/Gαs alone have little effect, but these two subthreshold signals combine to activate egg laying. We then transgenically expressed natural or designer GPCRs in the muscle cells and found that their subthreshold signals can also combine to induce muscle activity. However, artificially inducing strong signaling through just one of these GPCRs can be sufficient to induce egg laying. Knocking down Gαq and Gαs in the egg-laying muscle cells induced egg-laying defects that were stronger than those of a SER-1/SER-7 double knockout, indicating that additional endogenous GPCRs also activate the muscle cells. These results show that in the egg-laying muscles multiple GPCRs for serotonin and other signals each produce weak effects that individually do not result in strong behavioral outcomes. However, they combine to produce sufficient levels of Gαq and Gαs signaling to promote muscle activity and egg laying.SIGNIFICANCE STATEMENT How can neurons and other cells gather multiple independent pieces of information from the soup of chemical signals in their environment and compute an appropriate response? Most cells express >20 GPCRs that each receive one signal and transmit that information through three main types of G-proteins. We analyzed how this machinery generates responses by studying the egg-laying system of C. elegans, where serotonin and multiple other signals act through GPCRs on the egg-laying muscles to promote muscle activity and egg laying. We found that individual GPCRs within an intact animal each generate effects too weak to activate egg laying. However, combined signaling from multiple GPCR types reaches a threshold capable of activating the muscle cells.

Genetically encoded tools for in vivo G-protein-coupled receptor agonist detection at cellular resolution.

G-protein-coupled receptors (GPCRs) are the most abundant receptor type in the human body and are responsible for regulating many physiological processes, such as sensation, cognition, muscle contraction and metabolism. Further, GPCRs are widely expressed in the brain where their agonists make up a large number of neurotransmitters and neuromodulators. Due to the importance of GPCRs in human physiology, genetically encoded sensors have been engineered to detect GPCR agonists at cellular resolution in vivo. These sensors can be placed into two main categories: those that offer real-time information on the signalling dynamics of GPCR agonists and those that integrate the GPCR agonist signal into a permanent, quantifiable mark that can be used to detect GPCR agonist localisation in a large brain area. In this review, we discuss the various designs of real-time and integration sensors, their advantages and limitations, and some in vivo applications. We also discuss the potential of using real-time and integrator sensors together to identify neuronal circuits affected by endogenous GPCR agonists and perform detailed characterisations of the spatiotemporal dynamics of GPCR agonist release in those circuits. By using these sensors together, the overall knowledge of GPCR-mediated signalling can be expanded.

Identification of MRAP protein family as broad-spectrum GPCR modulators.

The melanocortin receptor accessory proteins (MRAP1 and MRAP2) are well-known endocrine regulators for the trafficking and signalling of all five melanocortin receptors (MC1R-MC5R). The observation of MRAP2 on regulating several non-melanocortin G protein-coupled receptors (GPCRs) has been sporadically reported, whereas other endogenous GPCR partners of the MRAP protein family are largely unknown. Here, we performed single-cell transcriptome analysis and drew a fine GPCR blueprint and MRAPs-associated network of two major endocrine organs, the hypothalamus and adrenal gland at single-cell resolution. We also integrated multiple bulk RNA-seq profiles and single-cell datasets of human and mouse tissues, and narrowed down a list of 48 GPCRs with strong endogenous co-expression correlation with MRAPs. 36 and 46 metabolic-related GPCRs were consequently identified as novel interacting partners of MRAP1 or MRAP2, respectively. MRAPs exhibited protein-protein interactions and varying pharmacological properties on the surface translocation, constitutive activities and ligand-stimulated downstream signalling of these GPCRs. Knockdown of MRAP2 expression by hypothalamic administration of adeno-associated virus (AAV)packed shRNA stimulated body weight gain in mouse model. Co-injection of corticotropinreleasing factor (CRF), the agonist of corticotropin releasing hormone receptor 1 (CRHR1), suppressed feeding behaviour in a MRAP2-dependent manner. Collectively, our study has comprehensively elucidated the complex GPCR networks in two major endocrine organs and redefined the MRAP protein family as broad-spectrum GPCR modulators. MRAP proteins not only serve as a vital endocrine pivot on the regulation of global GPCR activities in vivo that could explain the composite physiological phenotypes of the MRAP2 null murine model but also provide us with new insights of the phenotyping investigation of GPCR-MRAP functional complexes.

Luciferase-based GloSensor™ cAMP assay: Temperature optimization and application to cell-based kinetic studies.

G protein-coupled receptors (GPCRs) are an important receptor superfamily and common therapeutic targets. The second messenger cyclic adenosine monophosphate (cAMP) is a key mediator in many GPCR signaling pathways. Monitoring intracellular cAMP levels can help identify orthosteric agonists and antagonists, as well as allosteric modulators. In this regard, luminescence-based biosensors have revolutionized our ability to monitor GPCR signaling kinetics. The GloSensor™ cAMP assay enables real-time monitoring of signaling downstream of many GPCRs. However, it is crucial to optimize assay conditions such as temperature. As well, it has not been reported whether the effects of temperature on biosensor activity are reversible. Here, we describe the temperature sensitivity and reversibility of the GloSensor™ cAMP assay, and which GloSensor™ version is optimal for measuring cytosolic cAMP. We also present a detailed protocol for monitoring cAMP levels in live cells expressing endogenous or exogenous GPCRs. Temperature optimization studies were carried out using HEK293H cells transiently transfected with the adenosine receptor A2a and the GloSensor™ plasmid (pGloSensor-20F or -22F). We found that preincubation and luminescence reading at room temperature were optimal as compared to higher temperatures. As well, the GloSensor-22F biosensor had a superior signal-to-background ratio and the effect of temperature on biosensor activity was reversible. However, thermal instability of the biosensor may pose a problem for in vivo studies. Nevertheless, the GloSensor™ cAMP assay can be applied to analyze signaling by a wide range of GPCRs for drug discovery purposes.

Multiregional profiling of the brain transmembrane proteome uncovers novel regulators of depression.

Transmembrane proteins play vital roles in mediating synaptic transmission, plasticity, and homeostasis in the brain. However, these proteins, especially the G protein-coupled receptors (GPCRs), are underrepresented in most large-scale proteomic surveys. Here, we present a new proteomic approach aided by deep learning models for comprehensive profiling of transmembrane protein families in multiple mouse brain regions. Our multiregional proteome profiling highlights the considerable discrepancy between messenger RNA and protein distribution, especially for region-enriched GPCRs, and predicts an endogenous GPCR interaction network in the brain. Furthermore, our new approach reveals the transmembrane proteome remodeling landscape in the brain of a mouse depression model, which led to the identification of two previously unknown GPCR regulators of depressive-like behaviors. Our study provides an enabling technology and rich data resource to expand the understanding of transmembrane proteome organization and dynamics in the brain and accelerate the discovery of potential therapeutic targets for depression treatment.

Utilizing Designed Receptors Exclusively Activated by Designer Drug Chemogenetic Tools to Identify Beneficial G Protein-Coupled Receptor Signaling for Fibrosis.

Fibrosis or accumulation of extracellular matrix is an evolutionarily conserved mechanism adopted by an organism as a response to chronic injury. Excessive fibrosis, however, leads to disruption of organ homeostasis and is a common feature of many chronic diseases. G protein-coupled receptors (GPCRs) are important cell signaling mediators and represent molecular targets for many Food and Drug Administration-approved drugs. To identify new targets for fibrosis, we used a synthetic GPCR system named designed receptors exclusively activated by designer drugs (DREADDs) to probe signaling pathways essential for fibrotic response. We found that upon expression in human lung fibroblasts, activation of Gq- and Gs-DREADDs abrogated the induction of TGFβ-induced fibrosis marker genes. Genome-wide transcriptome analysis identified dysregulation of multiple GPCRs in lung fibroblasts treated with TGFβ To investigate endogenous GPCR modulating TGFβ signaling, we selected 13 GPCRs that signal through Gq or Gs and activated them by using specific agonists. We examined the impact of each agonist and how activation of endogenous GPCR affects TGFβ signaling. Among the agonists examined, prostaglandin receptor agonists demonstrated the strongest inhibitory effect on fibrosis. Together, we have demonstrated that the DREADDs system is a valuable tool to identify beneficial GPCR signaling for fibrosis. This study in fibroblasts has served as a proof of concept and allowed us to further develop in vivo models for fibrosis GPCR discovery. SIGNIFICANCE STATEMENT: Fibrosis is the hallmark of many end-stage cardiometabolic diseases, and there is an unmet medical need to discover new antifibrotic therapies, reduce disease progression, and bring clinically meaningful efficacy to patients. Our work utilizes designed receptors exclusively activated by designer drug chemogenetic tools to identify beneficial GPCR signaling for fibrosis, providing new insights into GPCR drug discovery.

Efforts Toward GPR88 Deorphanization: Challenges and Alternative Approaches to Troublesome Cell‐Based Second Messenger Assays

G protein‐coupled receptors (GPCR) comprise a family of over 800 integral membrane proteins involved in signal transduction and serve as the primary cellular sensors for chemical stimuli. About 35% of current drugs act on GPCR targets. In addition, of the 300 agents in current clinical trials, greater than 20% target novel GPCRs for which there are no approved drugs (orphan GPCRs). Ligand discovery and deorphanization efforts seeking to assign function to understudied GPCRs are not trivial. Notoriously complex in nature, GPCR‐dependent signaling centers around receptor‐ligand interactions that serve to stabilize conformational access to intracellular signaling partners, G proteins. In GPCR signaling, receptors and heterotrimeric G proteins (made of the Gα and Gβγ subunits) work together to transmit signals via downstream effectors and distinct pathways. The measurement of GPCR mediated second messengers such as cyclic AMP (cAMP), intracellular calcium (Ca2+) mobilization, and ERK activation (MAP kinase) are commonly used as tools to detect ligand induced GPCR activation. However, experimental pathway elucidation is complicated by the tendencies for pathway crosstalk, receptor desensitization, and receptor‐G protein interaction promiscuity. Cell‐based assays that monitor second messenger responses are often used as screening tools for potential GPCR agonists and antagonists, however, commonly used cell‐lines such as HEK‐293, express many endogenous GPCRs that can complicate data analysis. Many classes of Gα subunits exist: Gαs and Gα i/o G‐proteins modulate cyclic AMP (cAMP) through stimulation or inhibition of adenylate cyclase while Gαq subunits activate phospholipase C and a subsequent rapid release of intracellular Ca2+. Specifically, the orphan receptor, GPR88, has been linked with psychiatric diseases including schizophrenia and bipolar disorder. Lacking a validated endogenous agonist, the GPR88 signaling pathway remains poorly understood, however, in vivo knockout experiments and in vitro preclinical studies with small molecule synthetic agonists indicate that GPR88 couples to Gαi/o G‐proteins to inhibit adenylate cyclase activity and reduce intracellular cAMP in cell‐based assays. In this work, we describe our challenges in the study of the GPR88 signaling pathway using second messenger cell‐based assays and explain our decision to move to a real‐time NanoLuc‐based GPCR/G protein complementation assay.Support or Funding InformationNIH grant 1 R15 MH109034

Genetically Targeted Optical Control of an Endogenous G Protein-Coupled Receptor.

G protein-coupled receptors (GPCRs) are membrane proteins that play important roles in biology. However, our understanding of their function in complex living systems is limited because we lack tools that can target individual receptors with sufficient precision. State-of-the-art approaches, including DREADDs, optoXRs, and PORTL gated-receptors, control GPCR signaling with molecular, cell type, and temporal specificity. Nonetheless, these tools are based on engineered non-native proteins that may (i) express at nonphysiological levels, (ii) localize and turnover incorrectly, and/or (iii) fail to interact with endogenous partners. Alternatively, membrane-anchored ligands (t-toxins, DARTs) target endogenous receptors with molecular and cell type specificity but cannot be turned on and off. In this study, we used a combination of chemistry, biology, and light to control endogenous metabotropic glutamate receptor 2 (mGluR2), a Family C GPCR, in primary cortical neurons. mGluR2 was rapidly, reversibly, and selectively activated with photoswitchable glutamate tethered to a genetically targeted-plasma membrane anchor (membrane anchored Photoswitchable Orthogonal Remotely Tethered Ligand; maPORTL). Photoactivation was tuned by adjusting the length of the PORTL as well as the expression level and geometry of the membrane anchor. Our findings provide a template for controlling endogenous GPCRs with cell type specificity and high spatiotemporal precision.

Tango knock-ins visualize endogenous activity of G protein-coupled receptors in Drosophila

G protein-coupled receptors (GPCRs) represent a family of seven-pass transmembrane protein receptors whose ligands include neuropeptides and small-molecule neuromodulators such as dopamine and serotonin. These neurotransmitters act at long distances and are proposed to define the ground state of the nervous system. The Drosophila genome encodes approximately 50 neuropeptides and their functions in physiology and behavior are now under intensive studies. Key information currently lacking in the field is the spatiotemporal activation patterns of endogenous GPCRs. Here we report application of the Tango system, a reporter assay to detect GPCR activity, to endogenous GPCRs in the fly genome. We developed a method to integrate the sensor component of the Tango system to the C-terminus of endogenous genes by using genome editing techniques. We demonstrate that Tango sensors in the Sex-peptide receptor (SPR) locus allow sensitive detection of mating-dependent SPR activity in the female reproductive organ. The method is easily applicable to any GPCR and will provide a way to systematically characterize GPCRs in the fly brain.

Interrogating endogenous neuromodulatory GPCR signal processing by real‐time imaging of cAMP dynamics through intact neuronal circuits

The majority of neuromodulators exert their effects by activating G protein coupled receptors (GPCRs) which shape neuronal properties through the regulation of second messengers. The mechanisms of signal decoding at GPCRs amidst diversity of the effects associated with their ctivation remains elusive. Furthermore, there is a paucity of understanding of how GPCRs signal to produce unique responses impacting cellular physiology while converging on a limited set of second messenger signaling cascades. Although significant progress has been made studying GPCRs in reconstituted systems, information decoding processes by GPCRs in an endogenous setting is understood less due to a lack of appropriate tools. Here, we developed a novel in vivo reagent that allows monitoring GPCR signaling in the endogenous setting from genetically defined populations of cells. The approach takes advantage of knock‐in mice engineered to express a genetically encoded FRET‐based cAMP biosensor in a Cre‐dependent fashion. We applied this reagent to study integration of GPCR signals in striatal medium spiny neurons both in culture and in brain slices where intact circuits were activated by optogenetics. Combined with CRISPR/Cas9 genomic editing, this approach enabled us to identify individual receptors and analyze their contributions to downstream signaling in the native environment. We identified key spatial differences in pharmacological responsiveness by comparing dendritic regions with the neuronal cell body as well as plasma membrane and internal cellular signaling compartments. We then conducted comparative analysis of GPCR signaling mediated by two opposing circuits modulated by the neurotransmitter dopamine, revealing the mechanisms of dopaminergic processing as well as major differences in the generation of discrete second messenger signatures. We further demonstrate how cAMP signal processing mechanisms contribute to allostatic adaptations that imbalance tuning between striatal output neurons that may underlie states of reward and aversion.Support or Funding InformationNIH 1R01DK110621, APS STRIDE Undergraduate Summer Research Fellowship 1R25HL115473‐01.This work was supported by NIH grants: DA041207 (to B.S.M.), DA036596 (to K.A.M.), and DA026405 (to K.A.M.).