Subfamily Of G-protein Coupled Receptors Research Articles

Isoquinoline small molecule ligands are agonists and probe-dependent allosteric modulators of the glucagon subfamily of GPCRs

Class B1 G protein-coupled receptors (GPCRs) are peptide hormone receptors and well validated therapeutic targets, however development of non-peptide drugs targeting this class of receptors is challenging. Recently, a series of isoquinoline-based derivates were reported in the patent literature as allosteric ligands for the glucagon receptor subfamily, and two compounds, LSN3451217 and LSN3556672, were used to facilitate structural studies with the glucagon-like peptide-1 receptor (GLP-1R) and glucose dependent insulinotropic peptide receptor (GIPR) bound to orthosteric agonists. Here we pharmacologically characterized stereoisomers of LSN3451217 and LSN3556672, across the class B1 GPCR family. This revealed LSN3556672 isomers are agonists for the glucagon receptor (GCGR), GLP-1R, GIPR and the calcitonin receptor (CTR), albeit the degree of agonism varied at each receptor. In contrast, LSN3451217 isomers were more selective agonists at the GLP-1R, with lower potency at the GCGR and CTR and no activity at the GIPR. All compounds also modulated peptide-mediated cyclic adenosine monophosphate (cAMP) signaling at the GIPR, and to a lesser extent the GLP-1R, in a probe-dependent manner, with modest positive allosteric modulation observed for some peptides, and negligible effects observed with other peptides. In contrast neutral or weak negative/positive allosteric modulation was observed with peptides assessed at the GCGR and CTR. This study expands our knowledge on class B1 GPCR allosteric modulation and may have implications for future structural and drug discovery efforts targeting the class B1 GPCR subfamily.

Multiplexed mapping of the interactome of GPCRs with receptor activity-modifying proteins.

Receptor activity-modifying proteins (RAMPs) form complexes with G protein-coupled receptors (GPCRs) and may regulate their cellular trafficking and pharmacology. RAMP interactions have been identified for about 50 GPCRs, but only a few GPCR-RAMP complexes have been studied in detail. To elucidate a comprehensive GPCR-RAMP interactome, we created a library of 215 dual epitope-tagged (DuET) GPCRs representing all GPCR subfamilies and coexpressed each GPCR with each of the three RAMPs. Screening the GPCR-RAMP pairs with customized multiplexed suspension bead array (SBA) immunoassays, we identified 122 GPCRs that showed strong evidence for interaction with at least one RAMP. We screened for interactions in three cell lines and found 23 endogenously expressed GPCRs that formed complexes with RAMPs. Mapping the GPCR-RAMP interactome expands the current system-wide functional characterization of RAMP-interacting GPCRs to inform the design of selective therapeutics targeting GPCR-RAMP complexes.

Multiplexed selectivity screening of anti-GPCR antibodies.

G protein-coupled receptors (GPCRs) control critical cellular signaling pathways. Therapeutic agents including anti-GPCR antibodies (Abs) are being developed to modulate GPCR function. However, validating the selectivity of anti-GPCR Abs is challenging because of sequence similarities among individual receptors within GPCR subfamilies. To address this challenge, we developed a multiplexed immunoassay to test >400 anti-GPCR Abs from the Human Protein Atlas targeting a customized library of 215 expressed and solubilized GPCRs representing all GPCR subfamilies. We found that ~61% of Abs tested were selective for their intended target, ~11% bound off-target, and ~28% did not bind to any GPCR. Antigens of on-target Abs were, on average, significantly longer, more disordered, and less likely to be buried in the interior of the GPCR protein than the other Abs. These results provide important insights into the immunogenicity of GPCR epitopes and form a basis for designing therapeutic Abs and for detecting pathological auto-Abs against GPCRs.

Uncovering the functional role of GPR39 in the renal collecting duct

G protein-coupled receptors (GPCRs) are seven transmembrane domain receptors that comprise the largest class of proteins in the mammalian genome. GPR39 is part of the ghrelin subfamily of GPCRs, and is well-expressed in the kidney but has no reported function in renal physiology. To probe the renal role of GPR39, we first sought to localize it; in the absence of a validated immunofluorescence (IF) antibody, we used RNAscope. We found that Gpr39 localizes to nephron segments in the cortex and medulla which are positive for DBA (Dolichos Biflorus Agglutinin), a collecting duct marker. RNAScope expression of Gpr39 was highest in the inner medullary collecting duct, with lower levels in the outer medullary and cortical collecting ducts. Within the collecting duct, Gpr39 transcripts colocalized with Aqp2, indicating that Gpr39 is expressed specifically in principal cells. To address whether GPR39 is apical or basolateral, we cloned murine Gpr39 with a C-terminal EGFP tag and transiently transfected this construct into either polarized M-1 cells (murine collecting duct cell line) or polarized MDCK cells (Madin-Darby canine kidney cells); in both cases, cells were grown to confluence on permeable supports. In both cell lines, heterologous GPR39 trafficked to the basolateral membrane. Based on the localization of GPR39 to principal cells, we hypothesized that GPR39 activation may alter AQP2 expression or trafficking. To test this hypothesis in vitro, we first validated mpkCCD (mouse principal cell kidney cortical collecting duct) cells as a model. We cultured mpkCCD on transwells until polarized (TER >5kΩ·cm 2 ). In the absence of dDAVP, mpkCCD do not express AQP2 by western or IF. Basolateral treatment with 1nM dDAVP for 4 days induced AQP2 expression (western blot) and apical trafficking (confocal IF). We also confirmed that withdrawal of dDAVP (4h) resulted in a cytosolic accumulation of AQP2, while the re-introduction of dDAVP (1h) returned AQP2 to the apical membrane by IF. Moreover, we found that Gpr39 expression was unaffected by dDAVP by qPCR (n=3, ΔCt ± SEM: control = 7.9±0.3, dDAVP = 7.2±0.1) and western blot. In a preliminary study, we treated mpkCCD with 13nM of a GPR39 selective agonist, cpd1324, or with a vehicle control, and probed for changes in AQP2 by IF. Both groups were treated with 1nM dDAVP and with 10μM ZnCl2 (a GPR39 cofactor). We found that cpd1324 treatment decreased AQP2 by IF (mean integrated fluorescence density ± SEM (A.U.): cpd1324 = 233.5 ± 20.8, control = 373.8 ± 13.0). Additionally, cpd1324 treatment reduced the apical AQP2 abundance (mean integrated fluorescence density ± SEM (A.U.) of the most apical Z-slice: cpd1324 = 6.05e+05 ± 3.9e+04, control = 2.6e+06 ± 4.3e+05). These results suggest that GPR39 agonism may antagonize vasopressin-induced AQP2 trafficking and/or expression. NIH T32HL007534 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PsnGPCRdb: The Structure-network Database of G Protein Coupled Receptors

G protein coupled receptors (GPCRs) are critical eukaryotic signal transduction gatekeepers and represent the largest protein superfamily in the human proteome, with more than 800 members. They share seven transmembrane helices organized in an up-down bundle architecture. GPCR-mediated signaling pathways have been linked to numerous human diseases, and GPCRs are the targets of approximately 35% of all drugs currently on the market.Structure network analysis, a graph theory-based approach, represents a cutting-edge tool to deeply understand GPCR function, which strongly relies on communication between the extracellular and intracellular poles of their structure.psnGPCRdb stores the structure networks (i.e., linked nodes, hubs, communities and communication pathways) computed on all updated GPCR structures in the Protein Data Bank, in their isolated states or in complex with extracellular and/or intracellular molecules. The structure communication signatures of a sub-family or family of GPCRs as well as of their small-molecule activators or inhibitors are stored as consensus networks. The database stores also all meaningful structure network-based comparisons (i.e., difference networks) of functionally different states (i.e., inactive or active) of a given receptor sub-type, or of consensus networks representative of a receptor sub-type, type, sub-family or family. Single or consensus GPCR networks hold also information on amino acid conservation. The database allows to graphically analyze 3D structure networks together with interactive data-tables. Ligand-centric networks can be analyzed as well.psnGPCRdb is unique and represents a powerful resource to unravel GPCR function with important implications in cell signaling and drug design.psnGPCRdb is freely available at: http://webpsn.hpc.unimo.it/psngpcr.php.

GPR97 deficiency ameliorates renal interstitial fibrosis in mouse hypertensive nephropathy.

Hypertensive nephropathy (HTN) ranks as the second-leading cause of end-stage renal disease (ESRD). Accumulating evidence suggests that persistent hypertension injures tubular cells, leading to tubulointerstitial fibrosis (TIF), which is involved in the pathogenesis of HTN. G protein-coupled receptors (GPCRs) are implicated in many important pathological and physiological processes and act as important drug targets. In this study, we explored the intrarenal mechanisms underlying hypertension-associated TIF, and particularly, the potential role of GPR97, a member of the adhesion GPCR subfamily, in TIF. A deoxycorticosterone acetate (DOCA)/salt-induced hypertensive mouse model was used. We revealed a significantly upregulated expression of GPR97 in the kidneys, especially in renal tubules, of the hypertensive mice and 10 patients with biopsy-proven hypertensive kidney injury. GPR97-/- mice showed markedly elevated blood pressure, which was comparable to that of wild-type mice following DOCA/salt treatment, but dramatically ameliorated renal injury and TIF. In NRK-52E cells, we demonstrated that knockdown of GPR97 suppressed the activation of TGF-β signaling by disturbing small GTPase RhoA-mediated cytoskeletal reorganization, thus inhibiting clathrin-mediated endocytosis of TGF-β receptors and subsequent Smad activation. Collectively, this study demonstrates that GPR97 contributes to hypertension-associated TIF at least in part by facilitating TGF-β signaling, suggesting that GPR97 is a pivotal intrarenal factor for TIF progression under hypertensive conditions, and therapeutic strategies targeting GPR97 may improve the outcomes of patients with HTN.

Asymmetric activation of class C GPCRs.

Class C G-protein-coupled receptors (GPCRs) comprise a unique GPCR subfamily with large ligand-binding extracellular domains and function as obligate dimers. The recently resolved cryo-EM structures of full-length GABAB, CaSR, and mGlus have revealed that these receptors are activated in an asymmetric manner, leading to G-protein-coupling on one protomer within the receptor dimer. In this review we discuss the mechanisms of asymmetric activation in class C GPCRs and the unique mode of interaction with the inhibitory Gi protein. Upon activation, the two seven-transmembrane domains (7TMs) of class C GPCRs rearrange to form a conserved asymmetric TM6-TM6 interface. In contrast to class A and B GPCRs, G-protein coupling does not involve the cytoplasmic opening of TM6, but is facilitated through the coordination of intracellular loops. Furthermore, positive and negative allosteric modulators (PAMs and NAMs) adopt distinct conformations to regulate the activity of class C GPCRs. Taken together, these recent findings on the mechanism of asymmetric activation of class C GPCRs highlight a novel mechanism of G protein activation and provide new insights into the design of therapeutics targeting these receptors.

MUG: A mutation overview of GPCR subfamily A17 receptors

G protein-coupled receptors (GPCRs) mediate several signaling pathways through a general mechanism that involves their activation, upholding a chain of events that lead to the release of molecules responsible for cytoplasmic action and further regulation. These physiological functions can be severely altered by mutations in GPCR genes. GPCRs subfamily A17 (dopamine, serotonin, adrenergic and trace amine receptors) are directly related with neurodegenerative diseases, and as such it is crucial to explore known mutations on these systems and their impact in structure and function. A comprehensive and detailed computational framework - MUG (Mutations Understanding GPCRs) - was constructed, illustrating key reported mutations and their effect on receptors of the subfamily A17 of GPCRs. We explored the type of mutations occurring overall and in the different families of subfamily A17, as well their localization within the receptor and potential effects on receptor functionality. The mutated residues were further analyzed considering their pathogenicity. The results reveal a high diversity of mutations in the GPCR subfamily A17 structures, drawing attention to the considerable number of mutations in conserved residues and domains. Mutated residues were typically hydrophobic residues enriched at the ligand binding pocket and known activating microdomains, which may lead to disruption of receptor function. MUG as an interactive web application is available for the management and visualization of this dataset. We expect that this interactive database helps the exploration of GPCR mutations, their influence, and their familywise and receptor-specific effects, constituting the first step in elucidating their structures and molecules at the atomic level.

Olfactory Receptors as an Emerging Chemical Sensing Scaffold.

Chemical biosensors are an increasingly ubiquitous part of our lives. Beyond enzyme-coupled assays, recent synthetic biology advances now allow us to hijack more complex biosensing systems to respond to difficult to detect analytes, such as chemical small molecules. Here, we briefly overview recent advances in the biosensing of small molecules, including nucleic acid aptamers, allosteric transcription factors, and two-component systems. We then look more closely at a recently developed chemical sensing system, G protein-coupled receptor (GPCR)-based sensors. Finally, we consider the chemical sensing capabilities of the largest GPCR subfamily, olfactory receptors (ORs). We examine ORs' role in nature, their potential as a biomedical target, and their ability to detect compounds not amenable for detection using other biological scaffolds. We conclude by evaluating the current challenges, opportunities, and future applications of GPCR- and OR-based sensors.

Adhesion G protein-coupled receptor gluing action guides tissue development and disease.

Phylogenetic analysis of human G protein-coupled receptors (GPCRs) divides these transmembrane signaling proteins into five groups: glutamate, rhodopsin, adhesion, frizzled, and secretin families, commonly abbreviated as the GRAFS classification system. The adhesion GPCR (aGPCR) sub-family comprises 33 different receptors in humans. Majority of the aGPCRs are orphan receptors with unknown ligands, structures, and tissue expression profiles. They have a long N-terminal extracellular domain (ECD) with several adhesion sites similar to integrin receptors. Many aGPCRs undergo autoproteolysis at the GPCR proteolysis site (GPS), enclosed within the larger GPCR autoproteolysis inducing (GAIN) domain. Recent breakthroughs in aGPCR research have created new paradigms for understanding their roles in organogenesis. They play crucial roles in multiple aspects of organ development through cell signaling, intercellular adhesion, and cell-matrix associations. They are involved in essential physiological processes like regulation of cell polarity, mitotic spindle orientation, cell adhesion, and migration. Multiple aGPCRs have been associated with the development of the brain, musculoskeletal system, kidneys, cardiovascular system, hormone secretion, and regulation of immune functions. Since aGPCRs have crucial roles in tissue patterning and organogenesis, mutations in these receptors are often associated with diseases with loss of tissue integrity. Thus, aGPCRs include a group of enigmatic receptors with untapped potential for elucidating novel signaling pathways leading to drug discovery. We summarized the current knowledge on how aGPCRs play critical roles in organ development and discussed how aGPCR mutations/genetic variants cause diseases.

GPR68 Inhibition with a Novel Group of Ogremorphin Inhibitors Upregulate Endothelial Barrier Function and Protect Against Bacterial Pathogens or Acidosis‐induced Inflammation in Lung Endothelium

BackgroundEndothelial dysfunction characterized by an increase in endothelial permeability and inflammatory responses are two major pathological hallmarks of various lung disorders including the current global pandemic COVID‐19. Thus, drugs targeting the preservation and restoration of endothelial function represent an attractive therapeutic target to treat endothelial dysfunction‐derived cardiopulmonary diseases. G protein‐coupled receptors (GPCRs), especially a sub‐family of proton‐sensing GPCRs including GPR4 and GPR68, have been recently suggested to play a role in the modulation of endothelial function. In this study, we investigated the barrier protective and anti‐inflammatory effects of two recently developed novel class of GPR68 inhibitors: ogremorphins OGM8345 and OGM‐1 in cultured human lung endothelial cells as well as in mice.MethodsEndothelial permeability was measured by monitoring transendothelial electrical resistance (TER) across human pulmonary arterial endothelial cells (HPAECs). Protein and mRNA expression levels of endothelial inflammation markers were analyzed by western blot and quantitative real time PCR, respectively. Acidosis was induced by switching cells to acidic pH (6.5) medium and luciferase‐based Tango assay was performed to evaluate GPR68 activation. C57BL/6 mice were exposed to lipopolysaccharide (LPS from Escherichia coli) or heat‐killed Staphylococcus aureus (HKSA), and vascular leak/inflammation was assessed by determining the extravasation of intravenously injected Evans blue tracer into lungs and total cells/protein count in bronchoalveolar lavage samples.ResultsBoth OGM8345 (1‐5 µM) and OGM‐1 (0.3‐1.5 µM) induced a robust dose‐dependent increase in basal EC barrier function that was evident by an 150‐200% increase in TER values. Both inhibitors also markedly attenuated LPS‐ and HKSA‐induced endothelial hyperpermeability. LPS or HKSA‐induced upregulation of inflammatory cytokines/chemokines genes TNF‐α, ICAM‐1, VCAM‐1, IL‐6, IL‐8, IL‐1β, and CXCL5 was significantly attenuated by OGMs as demonstrated by RT‐PCR analysis. Consistently, both OGMs suppressed LPS‐ and HKSA‐induced protein expression of phospho‐NFkB, VCAM‐1 and ICAM‐1. In contrast, pharmacologic inhibition of GPR4 by NE 52‐QQ57 failed to alleviate LPS or HKSA‐induced EC barrier dysfunction and inflammation. Importantly, LPS, HKSA or acidosis stimulation resulted in increased GPR68 mRNA expression as well as GPR68 activity that was inhibited by OGMs. Both OGMs attenuated vascular leak and lung inflammation caused by intratracheal injection of LPS or HKSA in C57BL/6 mice as illustrated by reduced Evans blue accumulation in the lungs and significant inhibition of inflammatory cells and protein content in bronchoalveolar lavage samples.ConclusionAltogether, these results establish an essential role of GPR68 in endothelial dysfunction and strongly suggest a therapeutic potential of GPR68‐selective inhibitors in improving endothelial dysfunction‐derived lung injuries.

Genome-wide identification of 194 G protein-coupled receptor (GPCR) genes from the water flea Daphnia magna

In crustaceans, G protein–coupled receptors (GPCRs) are the largest transmembrane receptor family and function by mediating various environmental stimuli in cells. Understanding GPCR signaling is crucial to better understanding of crustacean endocrinology. GPCRs evolved from early eukaryotes, and genome-wide identification of GPCRs in a particular taxon can provide insight into evolutionary tendencies and adaptive strategies of GPCR response to environmental stimuli. Here, we identified 194 full-length GPCR genes in the water flea Daphnia magna that can be divided into five distinct classes (A, B, C, F, and other). A strong orthologous relationship for amine, neuropeptide, and opsin receptors was found in the phylogenetic comparison of D. magna GPCRs to those of humans and two well-known insects (Drosophila melanogaster and Solenopsis invicta). Our results based on phylogenetic relationships suggest that most GPCRs subfamilies have undergone sporadic evolutionary processes for adaptation to environmental pressures. Despite the dynamics of GPCR evolution, some GPCRs are highly conserved between species. This study provides a better understanding of the evolution of GPCRs and expands our knowledge of the potential physiological mechanisms in D. magna in response to various environmental stimuli.

Ligands and Beyond: Mechanosensitive Adhesion GPCRs.

Cells respond to diverse types of mechanical stimuli using a wide range of plasma membrane-associated mechanosensitive receptors to convert extracellular mechanical cues into intracellular signaling. G protein-coupled receptors (GPCRs) represent the largest cell surface protein superfamily that function as versatile sensors for a broad spectrum of bio/chemical messages. In recent years, accumulating evidence has shown that GPCRs can also engage in mechano-transduction. According to the GRAFS classification system of GPCRs, adhesion GPCRs (aGPCRs) constitute the second largest GPCR subfamily with a unique modular protein architecture and post-translational modification that are well adapted for mechanosensory functions. Here, we present a critical review of current evidence on mechanosensitive aGPCRs.

Classification Model for the Second Extracellular Loop of Class A GPCRs.

The extracellular loop 2 (ECL2) is the longest and the most diverse loop among class A G protein-coupled receptors (GPCRs). It connects the transmembrane (TM) helices 4 and 5 and contains a highly conserved cysteine through which it is bridged with TM3. In this paper, experimental ECL2 structures were analyzed based on their sequences, shapes, and intramolecular contacts. To take into account the flexibility, we incorporated into our analyses information from the molecular dynamics trajectories available on the GPCRmd website. Despite the high sequence variability, shapes of the analyzed structures, defined by the backbone volume overlaps, can be clustered into seven main groups. Conformational differences within the clusters can be then identified by intramolecular interactions with other GPCR structural domains. Overall, our work provides a reorganization of the structural information of the ECL2 of class A GPCR subfamilies, highlighting differences and similarities on sequence and conformation levels.

Constitutive, Basal, and β-Alanine-Mediated Activation of the Human Mas-Related G Protein-Coupled Receptor D Induces Release of the Inflammatory Cytokine IL-6 and Is Dependent on NF-κB Signaling.

G protein-coupled receptors (GPCRs) have emerged as key players in regulating (patho)physiological processes, including inflammation. Members of the Mas-related G protein coupled receptors (MRGPRs), a subfamily of GPCRs, are largely expressed by sensory neurons and known to modulate itch and pain. Several members of MRGPRs are also expressed in mast cells, macrophages, and in cardiovascular tissue, linking them to pseudo-allergic drug reactions and suggesting a pivotal role in the cardiovascular system. However, involvement of the human Mas-related G-protein coupled receptor D (MRGPRD) in the regulation of the inflammatory mediator interleukin 6 (IL-6) has not been demonstrated to date. By stimulating human MRGPRD-expressing HeLa cells with the agonist β-alanine, we observed a release of IL-6. β-alanine-induced signaling through MRGPRD was investigated further by probing downstream signaling effectors along the Gαq/Phospholipase C (PLC) pathway, which results in an IkB kinases (IKK)-mediated canonical activation of nuclear factor kappa-B (NF-κB) and stimulation of IL-6 release. This IL-6 release could be blocked by a Gαq inhibitor (YM-254890), an IKK complex inhibitor (IKK-16), and partly by a PLC inhibitor (U-73122). Additionally, we investigated the constitutive (ligand-independent) and basal activity of MRGPRD and concluded that the observed basal activity of MRGPRD is dependent on the presence of fetal bovine serum (FBS) in the culture medium. Consequently, the dynamic range for IL-6 detection as an assay for β-alanine-mediated activation of MRGPRD is substantially increased by culturing the cells in FBS free medium before treatment. Overall, the observation that MRGPRD mediates the release of IL-6 in an in vitro system, hints at a role as an inflammatory mediator and supports the notion that IL-6 can be used as a marker for MRGPRD activation in an in vitro drug screening assay.

Decoding Partner Specificity of Opioid Receptor Family.

This paper describes an exciting big data analysis compiled in a freely available database, which can be applied to characterize the coupling of different G-Protein coupled receptors (GPCRs) families with their intracellular partners. Opioid receptor (OR) family was used as case study in order to gain further insights into the physiological properties of these important drug targets, known to be associated with the opioid crisis, a huge socio-economic issue directly related to drug abuse. An extensive characterization of all members of the ORs family (μ (MOR), δ (DOR), κ (KOR), nociceptin (NOP)) and their corresponding binding partners (ARRs: Arr2, Arr3; G-protein: Gi1, Gi2, Gi3, Go, Gob, Gz, Gq, G11, G14, G15, G12, Gssh, Gslo) was carried out. A multi-step approach including models’ construction (multiple sequence alignment, homology modeling), complex assembling (protein complex refinement with HADDOCK and complex equilibration), and protein-protein interface characterization (including both structural and dynamics analysis) were performed. Our database can be easily applied to several GPCR sub-families, to determine the key structural and dynamical determinants involved in GPCR coupling selectivity.

Receptor Endocytosis as a Mechanism of Biased Agonism at CXCR3

G Protein Coupled Receptors (GPCRs) are the largest class of receptors in the human genome. They are expressed in virtually every cell type and are implicated in numerous physiological processes. GPCR signaling is highly specific due to biased agonism, in which different ligands, receptors, or cellular systems selectively activate different signaling pathways. The chemokine system, a subfamily of GPCRs primarily present on leukocytes, serves as an endogenous example of biased agonism due to the significant promiscuity between chemokine ligands and receptors. For example, the chemokine receptor CXCR3 and its ligands CXCL9, CXCL10, and CXCL11 have clearly demonstrated bias in vitro and in vivo. GPCRs are conventionally understood to signal exclusively from the plasma membrane. Therefore, the ligand-induced internalization of receptors is thought to desensitize G Protein-dependent signaling. However, recent research has shown that numerous GPCRs signal from intracellular membranes, including the endoplasmic reticulum, the golgi, and the endosome. This novel finding has led to the discovery that the subcellular location of a GPCR can affect its functional output. The objective of this study was to evaluate the contribution of receptor endocytosis to CXCR3's biased agonism. We hypothesize that CXCR3 signals from the early endosome, and as CXCL9, CXCL10, and CXCL11 promote different patterns of internalization, endocytic signaling is a mechanism for the functional selectivity of its endogenous ligands. We performed BRET and confocal microscopy on C-terminal phosphodeficient mutants of CXCR3 to measure their internalization in response to stimulation with CXCL9, CXCL10, or CXCL11. While stimulation with CXCL9 did not induce significant internalization of WT CXCR3 or any CXCR3 mutants, stimulation with CXCL10 or CXCL11 promoted β-arrestin mediated internalization of CXCR3 that was decreased in the phosphodeficient mutants. Internalization was almost completely eliminated at the CXCR3-L344X truncation mutant. We also measured the phosphorylation of extracellular-signal related kinase 1/2 (ERK 1/2) in response to stimulation with CXCL9, CXCL10, or CXCL11. These results showed that the CXCR3 mutants promoted more pERK 1/2 than WT CXCR3 when stimulated with CXCL10 or CXCL11. Notably, CXCR3-L344X promoted the most pERK 1/2 out of all studied receptors. This negative correlation between receptor internalization and pERK1/2 could suggest that endocytosis has an inhibitory effect on ERK signaling. To further assess the contribution of endocytic signaling to CXCR3's signaling effects, we will inhibit receptor endocytosis through treatment with Barbadin, a selective pharmacological inhibitor of the β-arrestin/AP2 complex required for endocytosis, or through overexpression of mutant dynamin, a protein responsible for scission of clathrin-coated endocytic vesicles. We will evaluate how inhibiting endocytosis impacts CXCR3's ligand-induced G Protein signal, ERK response, transcriptional reporter activation, and chemotaxis. We expect that inhibiting endocytosis will decrease the extent of bias between the three ligands, thus implicating location bias as a factor in CXCR3's functional selectivity.

Probing Allostery in Oligomeric M2 Receptors using smFRET

G Protein-Coupled Receptors (GPCRs) possess two types of ligand binding sites: orthosteric and allosteric sites. The highly conserved structure of the orthosteric sites results in low levels of ligand selectivity across members of a single GPCR subfamily, posing challenges for drug discovery. An alternative approach is the development of receptor-specific allosteric ligands, which modulate responsiveness of GPCRs to orthosteric ligands. There is still an open question whether some GPCRs function as monomers, dimers or higher oligomers, in particular regarding cooperative interactions between orthosteric and allosteric sites in different units. Here we used the M2 muscarinic receptor (M2R) to quantify these inter-monomeric interactions using single-molecule Förster Resonance energy Transfer (smFRET). Oligomeric preparations of M2R have been designed to contain receptors with vacant orthosteric sites and intact allosteric sites. The smFRET construct added a small peptide sequence at the N-terminal (ybbR-tag) and a tetra-cysteine sequence the extracellular loop 2 (ECL2) (tetra-cysteine), which specifically bind to Co-enzyme a (CoA) conjugated fluorophores (AlexaFluoro®647, AF647), and the Fluorescein Arsenical Hairpin (FlAsH), respectively. To obtain a high-yield of fluorescently labelled receptors, detergent-based preparations of M2R were expressed and purified from suspension-based Expi-CHO cells. Immunoblotting analysis of purified M2R under non-denaturing conditions revealed that the receptor preparations are predominantly oligomeric in nature. Labelling of M2R with FlAsH and CoA-AF647 was confirmed using a series of steady-state and time-resolved fluorescence-based assays. Using the designed sensor, smFRET measurements were performed on monomeric M2 controls and on different oligomeric samples in the presence/absence of allosteric modulators. The data confirms the presence of allosteric interactions between constituent protomers within oligomers of M2, both in vitro and in live cells.

Ligand Docking Methods to Recognize Allosteric Inhibitors for G-Protein-Coupled Receptors

G-protein-coupled receptors (GPCRs) are membrane proteins which play an important role in many cellular processes and are excellent drug targets. Despite the existence of several US Food and Drug Administration (FDA)-approved GPCR-targeting drugs, there is a continuing challenge of side effects owing to the nonspecific nature of drug binding. We have investigated the diversity of the ligand binding site for this class of proteins against their cognate ligands using computational docking, even if their structures are known already in the ligand-complexed form. The cognate ligand of some of these receptors dock at allosteric binding site with better score than the binding at the conservative site. Interestingly, amino acid residues at such allosteric binding site are not conserved across GPCR subfamilies. Such a computational approach can assist in the prediction of specific allosteric binders for GPCRs.

Abstract P3-06-07: ADGRF1 overexpression inhibits tumor growth in vivo by inducing cell cycle arrest in HER2+ breast cancer

Abstract Background: Adhesion G protein-coupled receptors (GPCRs) form the second largest GPCR subfamily and control many cellular processes involved in cancer. ADGRF1 is an adhesion GPCR overexpressed and gene amplified in HER2+ and triple-negative breast cancer (BC). We have previously found a role of ADGRF1 in promoting tumorigenicity and metastatic processes in cell-based studies in HER2+ BC. The goal of this study was to evaluate the effects of ADGRF1 overexpression in vivo in HER2+ BC. Methods: BT474 stable cell lines were generated using lentiviral system containing ADGRF1 cDNA using PHAGE system, overexpressing ADGRF1 in Doxycycline (Dox)-inducible manner. Mice (N=8/group) were injected with various numbers of cells with (+) or without (-) Dox (10K, 30K, 100K, 300K, 1 million) to assess for tumor initiation and growth. Transcriptomic and proteomic analysis of ADGRF1-overexpressing BT474 cells was also performed using RNAseq and RPPA analysis, respectively. The candidates were validated using western blotting and IHC staining. Cell cycle analysis was conducted by measuring DNA content with propidium iodide using flow cytometry. Results: Dox-induced overexpression of ADGRF1 in mice led to inhibition of tumor growth compared to no Dox treated tumors. Furthermore, fewer mice injected with Dox-treated cells formed tumors. Our RNA-Seq data revealed downregulation of genes involved in cell cycle regulation, which included E2F targets, G2M checkpoint pathways, and MYC targets. Likewise in RPPA analysis, several proteins in the cell cycle pathway (FOXO1, AuroraA, Ki67, Ezh2, CHAF1, DEPTOR, Rb1, Phospho-Rb (S780), and Hexokinase II) were downregulated by ADGRF1 overexpression. Validation of RNAseq and RPPA candidates demonstrated lower number of Ki67+ cells and reduced NF-kB expression, and inhibition of STAT3 phosphorylation. Furthermore, we found that ADGRF1 overexpression by Dox also led to cell cycle arrest at GO/G1 phase. Conclusion: Our results suggest that ADGRF1 overexpression inhibits tumor growth in vivo by inducing cell cycle arrest in HER2+ breast cancer. These results are in contrast to the in vitro data demonstrating a role of ADGRF1 in promoting tumorigenicity and metastatic processes in HER2+ BC. Understanding the mechanism of this difference is critical in investigating ADGRF1 as a drug target in breast cancer. Citation Format: Raksha Bhat, Noor Mazin Abdulkareem, Lanfang Qin, Carmine De Angelis, Hala Yasser, Hariprasad Thangavel, Jennifer Torres, Vidyalakshmi Sethunath, Tamika Mitchell, Sarmistha Nanda, Resel Pereira, Martin Shea, Rachel Schiff, Meghana Trivedi. ADGRF1 overexpression inhibits tumor growth in vivo by inducing cell cycle arrest in HER2+ breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-07.