August 2022 "Fluorescently labeled ligands are versatile molecular tools to study G protein-coupled receptors (GPCRs) and can be used for a range of different applications, including bioluminescence resonance energy transfer (BRET) assays. Here, we report the structure-based development of fluorescent ligands targeting the intracellular allosteric binding site (IABS) of the CC chemokine receptor 2 (CCR2), a class A GPCR that has been pursued as a drug target in oncology and inflammation. Starting from previously reported intracellular CCR2 antagonists, several tetramethylrhodamine (TAMRA)-labeled CCR2 ligands were designed, synthesized, and tested for their suitability as fluorescent reporters to probe binding to the IABS of CCR2. By means of these studies, we developed 14 as a fluorescent CCR2 ligand, enabling cell-free as well as cellular NanoBRET-based binding studies in a non-isotopic and high-throughput manner. Further, we show that 14 can be used as a tool for fragment-based screening approaches. Thus, our small-molecule-based fluorescent CCR2 ligand 14 represents a promising tool for future studies of CCR2 pharmacology." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "Background and purpose: Misuse of opioids has greatly affected our society. One potential solution is to develop analgesics that act at targets other than opioid receptors. These can be used either as stand-alone therapeutics or to improve the safety profile of opioid drugs. Previous research showed that activation of Gq/11 proteins by G-protein coupled receptors has pro-nociceptive properties, suggesting that blockade of Gq/11 signalling could be beneficial for pain control. The aim of this study was to test this hypothesis pharmacologically by using potent and selective Gq/11 inhibitor YM-254890. Experimental approach: We used a series of behavioural assays to evaluate the acute responses of mice to painful thermal stimulation while administering YM-254890 alone and in combination with morphine. We then used electrophysiological recordings to evaluate the effects of YM-254890 on the excitability of dorsal root ganglion (DRG) nociceptor neurons." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 A NanoBRET-Based H 3 R Conformational Biosensor to Study Real-Time H 3 Receptor Pharmacology in Cell Membranes and Living Cells "Conformational biosensors to monitor the activation state of G protein-coupled receptors are a useful addition to the molecular pharmacology assay toolbox to characterize ligand efficacy at the level of receptor proteins instead of downstream signaling. We recently reported the initial characterization of a NanoBRET-based conformational histamine H3 receptor (H3R) biosensor that allowed the detection of both (partial) agonism and inverse agonism on living cells in a microplate reader assay format upon stimulation with H3R ligands. In the current study, we have further characterized this H3R biosensor on intact cells by monitoring the effect of consecutive ligand injections in time and evaluating its compatibility with photopharmacological ligands that contain a light-sensitive azobenzene moiety for photo-switching. In addition, we have validated the H3R biosensor in membrane preparations and found that observed potency values better correlated with binding affinity values that were measured in radioligand competition binding assays on membranes. Hence, the H3R conformational biosensor in membranes might be a ready-to-use, high-throughput alternative for radioligand binding assays that in addition can also detect ligand efficacies with comparable values as the intact cell assay." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 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" Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "Genetically encoded fluorescent biosensors allow intracellular signaling dynamics to be tracked in live cells and tissues using optical detection. Many such biosensors are based on the principle of Förster resonance energy transfer (FRET), and we have recently developed a simple approach for in vivo detection of FRET-based biosensor signals using fiber photometry. By combining fiber photometry with FRET-based biosensors, we were able to track GPCR-dependent signaling pathways over time, and in response to drug treatments in freely-moving adult rats. Recording from specific neuronal populations, we can quantify intracellular signaling while simultaneously measuring behavioral responses. Our approach, described in detail here, uses adeno-associated viruses infused intracerebrally in order to express genetically-encoded FRET-based biosensors. After several weeks to allow biosensor expression, fiber photometry is used in order to record drug responses in real time from freely-moving adult rats. This methodology would be compatible with other mammalian species and with many biosensors. Hence, it has wide applicability across a spectrum of neuroscience research, ranging from the study of neural circuits and behavior, to preclinical drug development and screening." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 GPR3 expression in retinal ganglion cells contributes to neuron survival and accelerates axonal regeneration after optic nerve crush in mice "Glaucoma is an optic neuropathy and is currently one of the most common diseases that leads to irreversible blindness. The axonal degeneration that occurs before retinal ganglion neuronal loss is suggested to be involved in the pathogenesis of glaucoma. G protein-coupled receptor 3 (GPR3) belongs to the class A rhodopsin-type GPCR family and is highly expressed in various neurons. GPR3 is unique in its ability to constitutively activate the Gαs protein without a ligand, which elevates the basal intracellular cAMP level. Our earlier reports suggested that GPR3 enhances both neurite outgrowth and neuronal survival. However, the potential role of GPR3 in axonal regeneration after neuronal injury has not been elucidated. Herein, we investigated retinal GPR3 expression and its possible involvement in axonal regeneration after retinal injury in mice. GPR3 was relatively highly expressed in retinal ganglion cells (RGCs). Surprisingly, RGCs in GPR3 knockout mice were vulnerable to neural death during aging without affecting high intraocular pressure (IOP) and under ischemic conditions. Primary cultured neurons from the retina showed that GPR3 expression was correlated with neurite outgrowth and neuronal survival. Evaluation of the effect of GPR3 on axonal regeneration using GPR3 knockout mice revealed that GPR3 in RGCs participates in axonal regeneration after optic nerve crush (ONC) under zymosan stimulation. In addition, regenerating axons were further stimulated when GPR3 was upregulated in RGCs, and the effect was further augmented when combined with zymosan treatment. These results suggest that GPR3 expression in RGCs helps maintain neuronal survival and accelerates axonal regeneration after ONC in mice." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "Several G-protein coupled receptors (GPCR) are upregulated in Alzheimer's Disease (AD), which ought to facilitate neurotransmission, and improve cognition. Yet, despite this upregulation, associated physiological effects are not observed in AD patients. This paradox solicits urgent attention to find a suitable justification for disturbed neurotransmission in AD. This article focuses on the role of Aβ granules and their possible interaction with GPCRs that modulate neurotransmission and AD progression." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "Background and purpose: Vascular tone is regulated by the relative contractile state of vascular smooth muscle cells (VSMCs). Several integrins directly modulate VSMC contraction by regulating calcium influx through L-type voltage-gated Ca2+ channels (VGCCs). Genetic variants in ITGA9, which encodes the α9 subunit of integrin α9β1, and SVEP1, a ligand for integrin α9β1, associate with elevated blood pressure; however, neither SVEP1 nor integrin α9β1 has reported roles in vasoregulation. We determined whether SVEP1 and integrin α9β1 can regulate VSMC contraction." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 G protein-coupled receptor kinase type 2 and β-arrestin2: Key players in immune cell functions and inflammation "G protein-coupled receptor kinase type 2 (GRK2) and β-arrestin2 are representative proteins that regulate the transduction and trafficking of G protein-coupled receptor (GPCR) signaling. The kinase GRK2 and the multifunctional scaffolding protein β-arrestin2 are key integrated signaling nodes in various biological processes, and both of them regulate cell proliferation and promote cell invasion and migration. GRK2/β-arrestin2 play multiple roles in the pathological mechanisms of a wide range of diseases including heart failure, cancer, and inflammatory diseases. This review summarizes the roles of GRK2/β-arrestin2 in immune cell function and focuses on the pathological implications of GRK2/β-arrestin2 in various inflammatory diseases." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "Bone remodeling is precisely regulated mainly by osteoblasts and osteoclasts. Although some G-protein coupled receptors (GPCRs) were reported to play roles in osteoblast function, little is known about the roles in osteoclasts. In this study, we found, for the first time, that the expression of GPR110 increased during osteoclastogenesis. GPR110 belongs to adhesion GPCR and was the functional receptor of N-docosahexaenoyl ethanolamine (also called synaptamide). Synaptamide suppressed osteoclastogenesis induced by receptor activator of nuclear factor-kappa B ligand. Considering that synaptamide is the endogenous metabolite of DHA, we hypothesized that DHA may inhibit osteoclastogenesis by affecting synaptamide/GPR110 signaling. But GPR110 knockout and subsequent rescue experiments revealed a pivotal role of GPR110 in the attenuation of osteoclastogenesis by synaptamide but not by DHA. These results suggest that synaptamide/GPR110 signaling negatively regulates osteoclastogenesis. Our study suggested that ligands of GPR110, such as synaptamide, might be a useful drug for osteoporotic patients." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "Aims: The pathophysiological mechanism(s) underlying non-alcoholic fatty liver disease (NAFLD) have yet to be fully delineated and only a single drug, peroxisome proliferator-activated receptor (PPAR) α/γ agonist saroglitazar, has been approved. Here, we sought to investigate the role of Regulator of G Protein Signaling 7 (RGS7) in hyperlipidemia-dependent hepatic dysfunction. " Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 TAS2R supports odontoblastic differentiation of human dental pulp stem cells in the inflammatory microenvironment "Background: Inflammatory microenvironment promotes odontoblastic differentiation in human dental pulp stem cells (hDPSCs), but the regulatory mechanisms remain unclear. In this study, we aimed to explore the role of TAS2R in odontoblastic differentiation of hDPSCs in the inflammatory microenvironment. Methods: Microarray analysis was performed to explore the differential mRNA profiles in inflammatory and healthy pulp tissues from the patients. hDPSCs isolated from the healthy pulp tissues were stimulated by LPS, TNFα and IL-6, respectively, to verify the effect of TAS2R. The expression markers related to odontoblastic differentiation of hDPSCs were observed by qPCR and chemical staining methods. TAS2R10 was overexpressed or silenced to observe the effect on odontoblastic differentiation of hDPSCs under LPS stimulation. The G protein and intracellular Ca2+ were detected, respectively, by qPCR and Fluo-4AM Ca2+ fluorescent probe." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "Chemokines, a subgroup of cytokines along with their receptors, are involved in various biologic processes and regulation of a wide range of immune responses in different physiologic and pathologic states such as tissue repair, infection, and inflammation. C-X-C motif chemokine receptor 4 (CXCR4), a G-protein-coupled receptor (GPCR), has one identified natural ligand termed stromal-derived factor-1(SDF-1 or CXCL12). Evidence demonstrated that the ligation of SDF-1 to CXCR4 initiates several intracellular signaling pathways, regulating cell proliferation, survival, chemotaxis, migration, angiogenesis, adhesion, as well as bone marrow (BM)-resident cells homing and mobilization. Additionally, CXCR4 is expressed by tumor cells in blood malignancies and solid tumors." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 A correlation study of adhesion G protein-coupled receptors as potential therapeutic targets in Uterine Corpus Endometrial cancer "Adhesion G protein-coupled receptors (adhesion GPCRs), as a member of the G protein-coupled receptors (GPCRs) superfamily, have gradually entered the field of vision of researchers. The structure, function, and involvement of adhesion GPCRs in cancer development have been discussed in a series of papers. Uterine Corpus Endometrial Carcinoma (UCEC) isa malignanttumorofendometrium epithelial, whichis alsooneofthemostcommonfemalereproductivesystemtumors, but there are few pieces of research related to adhesion GPCRs in UCEC." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "GPR21 is an orphan and constitutively active receptor belonging to the superfamily of G-Protein Coupled Receptors (GPCRs). GPR21 couples to the Gq family of G proteins and is expressed in macrophages. Studies of GPR21 knock-out mice indicated that GPR21 may be involved in promoting macrophage migration. The aim of this study was to evaluate the role of GPR21 in human macrophages, analyzing (i) its involvement in cell migration and cytokine release and (ii) the consequence of its pharmacological inhibition by using the inverse agonist GRA2. THP-1 cells were activated and differentiated into either M1 or M2 macrophages. GPR21 expression was evaluated at gene and protein level, the signalling pathway was investigated by an IP1 assay, and cytokine release by ELISA. Cell migration was detected by the Boyden chamber migration assay, performed on macrophages derived from both the THP-1 cell line and human peripheral blood monocytes." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "Histamine exerts its physiological functions through its four receptor subtypes. In this work, we report the subcellular localization of histamine receptor 2 (H2R), a G protein-coupled receptor (GPCR), which is expressed in a wide variety of cell and tissue types. A growing number of GPCRs have been shown to be localized in the nucleus and contribute toward transcriptional regulation. In this study, for the first time, we demonstrate the nuclear localization of H2R in lymphatic endothelial cells. In the presence of its ligand, we show significant upregulation of H2R nuclear translocation kinetics. Using fluorescently tagged histamine, we explored H2R-histamine binding interaction, which exhibits a critical role in this translocation event. Altogether, our results highlight the previously unrecognized nuclear localization pattern of H2R. At the same time, H2R as a GPCR imparts many unresolved questions, such as the functional relevance of this localization, and whether H2R can contribute directly to transcriptional regulation and can affect lymphatic specific gene expression. H2R blockers are commonly used medications that recently have shown significant side effects. Therefore, it is imperative to understand the precise molecular mechanism of H2R biology. In this aspect, our present data shed new light on the unexplored H2R signaling mechanisms. This article has an associated First Person interview with the first author of the paper." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "Oxysterols induce the migration of B-lymphocytes and dendritic cells to interfollicular regions of lymphoid tissues through binding the EBI2 (GPR183) to stimulate effective adaptive immunity and antibody production during infection. Aberrant EBI2 signaling is implicated in inflammatory bowel disease, sclerosis, and infectious disease. Here, we report the cryo-EM structures of an EBI2-Gi signaling complex with its endogenous agonist 7α,25-OHC and that of an inactive EBI2 bound to the inverse agonist GSK682753A. These structures reveal an agonist binding site for the oxysterol and a potential ligand entrance site exposed to the lipid bilayer. Mutations within the oxysterol binding site and the Gαi interface attenuate G protein signaling and abolish oxysterol-mediated cell migration indicating that G protein signaling directly involves in the oxysterol-EBI2 pathway. Together, these findings provide new insight into how EBI2 is activated by an oxysterol ligand and will facilitate the development of therapeutic approaches that target EBI2-linked diseases." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "Host response to infection involves the activation of the complement system leading to the production of anaphylatoxins C3a and C5a. Complement factor C5a exerts its effect through the activation of C5aR1, chemotactic receptor 1, and triggers the G protein-coupled signaling cascade. Orthosteric and allosteric antagonists of C5aR1 are a novel strategy for anti-inflammatory therapies. Here, we discuss recent crystal structures of inactive C5aR1 in terms of an inverted orientation of helix H8, unobserved in other GPCR structures. An analysis of mutual interactions of subunits in the C5aR1-G protein complex has provided new insights into the activation mechanism of this distinct receptor. By comparing two C5aR receptors C5aR1 and C5aR2 we explained differences between their signaling pathways on the molecular level. By means of molecular dynamics we explained why C5aR2 cannot transduce signal through the G protein pathway but instead recruits beta-arrestin. A comparison of microsecond MD trajectories started from active and inactive C5aR1 receptor conformations has provided insights into details of local and global changes in the transmembrane domain induced by interactions with the Gα subunit and explained the impact of inverted H8 on the C5aR1 activation." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "G-protein-coupled receptors (GPCR) are present at the cell surface in different conformational and oligomeric states. However, how these states impact GPCRs biological function and therapeutic targeting remains incompletely known. Here, we investigated this issue in living cells for the CC chemokine receptor 5 (CCR5), a major receptor in inflammation and the principal entry co-receptor for Human Immunodeficiency Viruses type 1 (HIV-1). We used TIRF microscopy and a statistical method to track and classify the motion of different receptor subpopulations. We showed a diversity of ligand-free forms of CCR5 at the cell surface constituted of various oligomeric states and exhibiting transient Brownian and restricted motions. These forms were stabilized differently by distinct ligands. In particular, agonist stimulation restricted the mobility of CCR5 and led to its clustering, a feature depending on β-arrestin, while inverse agonist stimulation exhibited the opposite effect. These results suggest a link between receptor activation and immobilization. Applied to HIV-1 envelope glycoproteins gp120, our quantitative analysis revealed agonist-like properties of gp120s. " Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "G-protein-coupled receptors (GPCRs) receive signals from ligands with different efficacies, and transduce to heterotrimeric G-proteins to generate different degrees of physiological responses. Previous studies revealed how ligands with different efficacies activate GPCRs. Here, we investigate how a GPCR activates G-proteins upon binding ligands with different efficacies. We report the cryo-EM structures of β1-adrenergic receptor (β1-AR) in complex with Gs (GαsGβ1Gγ2) and a partial agonist or a very weak partial agonist, and compare them to the β1-AR-Gs structure in complex with a full agonist. Analyses reveal similar overall complex architecture, with local conformational differences. Cellular functional studies with mutations of β1-AR residues show effects on the cellular signaling from β1-AR to the cAMP response initiated by the three different ligands, with residue-specific functional differences. Biochemical investigations uncover that the intermediate state complex comprising β1-AR and nucleotide-free Gs is more stable when binding a full agonist than a partial agonist. Molecular dynamics simulations support the local conformational flexibilities and different stabilities among the three complexes. These data provide insights into the ligand efficacy in the activation of GPCRs and G-proteins." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "Single particle cryogenic-electron microscopy (cryo-EM) is used extensively to determine structures of activated G protein-coupled receptors (GPCRs) in complex with G proteins or arrestins. However, applying it to GPCRs without signaling proteins remains challenging because most receptors lack structural features in their soluble domains to facilitate image alignment. In GPCR crystallography, inserting a fusion protein between transmembrane helices 5 and 6 is a highly successful strategy for crystallization. Although a similar strategy has the potential to broadly facilitate cryo-EM structure determination of GPCRs alone without signaling protein, the critical determinants that make this approach successful are not yet clear. Here, we address this shortcoming by exploring different fusion protein designs, which lead to structures of antagonist bound A2A adenosine receptor at 3.4 Å resolution and unliganded Smoothened at 3.7 Å resolution. The fusion strategies explored here are likely applicable to cryo-EM interrogation of other GPCRs and small integral membrane proteins." Read more at the source #DrGPCR #GPCR #IndustryNews

"Protecting neurons from death during oxidative and neuroexcitotoxic stress is key for preventing cognitive dysfunction. We uncovered a novel neuroprotective mechanism involving interaction between neurotrophic factor-α1 (NF-α1/carboxypeptidase E, CPE) and human 5-HTR1E, a G protein-coupled serotonin receptor with no previously known neurological function. Co-immunoprecipitation and pull-down assays confirmed interaction between NFα1/CPE and 5-HTR1E and 125I NF-α1/CPE-binding studies demonstrated saturable, high-affinity binding to 5-HTR1E in stably transfected HEK293 cells (Kd = 13.82 nM). Treatment of 5-HTR1E stable cells with NF-α1/CPE increased pERK 1/2 and pCREB levels which prevented a decrease in pro-survival protein, BCL2, during H2O2-induced oxidative stress. Cell survival assay in β-arrestin Knockout HEK293 cells showed that the NF-α1/CPE-5-HTR1E-mediated protection against oxidative stress was β-arrestin-dependent. Molecular dynamics studies revealed that NF-α1/CPE interacts with 5-HTR1E via 3 salt bridges, stabilized by several hydrogen bonds, independent of the serotonin pocket. Furthermore, after phosphorylating the C-terminal tail and intracellular loop 3 (ICL3) of NF-α1/CPE-5-HTR1E, it recruited β-arrestin1 by forming numerous salt bridges and hydrogen bonds to ICL2 and ICL3, leading to activation of β-arrestin1. Immunofluorescence studies showed 5-HTR1E and NF-α1/CPE are highly expressed and co-localized on cell surface of human hippocampal neurons. Importantly, knock-down of 5-HTR1E in human primary neurons diminished the NF-α1/CPE-mediated protection of these neurons against oxidative stress and glutamate neurotoxicity-induced cell death. Thus, NF-α1/CPE uniquely interacts with serotonin receptor 5-HTR1E to activate the β-arrestin/ERK/CREB/BCL2 pathway to mediate stress-induced neuroprotection." Read full article

August 2022 GRK2 selectively attenuates the neutrophil NADPH-oxidase response triggered by β-arrestin recruiting GPR84 agonists "In order to avoid a prolonged pro-inflammatory neutrophil response, signaling downstream of an agonist-activated G protein-coupled receptor (GPCR) has to be rapidly terminated. Among the family of GPCR kinases (GRKs) that regulate receptor phosphorylation and signaling termination, GRK2, which is highly expressed by immune cells, plays an important role. The medium chain fatty acid receptor GPR84 as well as formyl peptide receptor 2 (FPR2), receptors expressed in neutrophils, play a key role in regulating inflammation. In this study, we investigated the effects of GRK2 inhibitors on neutrophil functions induced by GPR84 and FPR2 agonists. GRK2 was shown to be expressed in human neutrophils and analysis of subcellular fractions revealed a cytosolic localization. " Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "Epithelial tube formation requires Rho1-dependent actomyosin contractility to generate the cellular forces that drive cell shape changes and rearrangement. Rho1 signaling is activated by G-protein-coupled receptor (GPCR) signaling at the cell surface. During Drosophila embryonic salivary gland (SG) invagination, the GPCR ligand Folded gastrulation (Fog) activates Rho1 signaling to drive apical constriction. The SG receptor that transduces the Fog signal into Rho1-dependent myosin activation has not been identified. Here, we reveal that the Smog GPCR transduces Fog signal to regulate Rho kinase accumulation and myosin activation in the medioapical region of cells to control apical constriction during SG invagination. We also report on unexpected Fog-independent roles for Smog in maintaining epithelial integrity and organizing cortical actin. Our data support a model wherein Smog regulates distinct myosin pools and actin cytoskeleton in a ligand-dependent manner during epithelial tube formation." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "Emerging evidence suggests that G protein-coupled receptor (GPCR) kinases (GRKs) are associated with the pathophysiology of Alzheimer's disease (AD). However, GRKs have not been directly implicated in regulation of the amyloid-β (Aβ) pathogenic cascade in AD. Here, we determine that GRKs phosphorylate a non-canonical substrate, anterior pharynx-defective 1A (APH1A), an integral component of the γ-secretase complex. Significantly, we show that GRKs generate distinct phosphorylation barcodes in intracellular loop 2 (ICL2) and the C terminus of APH1A, which differentially regulate recruitment of the scaffolding protein β-arrestin 2 (βarr2) to APH1A and γ-secretase-mediated Aβ generation. Further molecular dynamics simulation studies reveal an interaction between the βarr2 finger loop domain and ICL2 and ICL3 of APH1A, similar to a GPCR-β-arrestin complex, which regulates γ-secretase activity. Collectively, these studies provide insight into the molecular and structural determinants of the APH1A-βarr2 interaction that critically regulate Aβ generation." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "The X-linked form of Kallmann syndrome (KS), characterized by hypogonadotropic hypogonadism and anosmia, is due to mutations in the ANOS1 gene that encodes for the extracellular matrix (ECM) protein anosmin 1. Prokineticins (PKs) exert their biological functions through the activation of the G protein-coupled receptors (GPCRs) prokineticin receptor 1 and 2 (PKR1, 2), and mutations in the PK2 and PKR2 genes are involved in the pathogenesis of KS. We have previously shown interaction between PKR2 and anosmin 1 in vitro. In the current report we present evidence of the modulation of PK2/PKR2 activity by anosmin 1, since this protein is able to enhance the activation of the ERK1/2 (extracellular signal-regulated kinase 1/2) pathway elicited by PK2 through PKR2. We also show that the N-terminal region of anosmin 1, capable of binding to the PK2-binding domain of PKR2, seems to be responsible for this effect. The whey acidic protein domain (WAP) is necessary for this modulatory activity, although data from GST pull-down (glutathione-S-transferase) and analysis of the N267K mutation in the fibronectin type III domain 1 (FnIII.1) suggest the cysteine-rich (CR) and the FnIII.1 domains could assist the WAP domain both in the binding to PKR2 and in the modulation of the activation of the receptor by PK2. Our data support the idea of a modulatory role of anosmin 1 in the biological effects controlled by the PK2/PKR2 system." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "Background: Activation of signaling effectors by G-protein coupled receptors (GPCRs) depends on different molecular mechanisms triggered by conserved amino acid residues. Although studies have focused on the G-protein signaling state, the mechanism for β-arrestin signaling by CB1 is not yet well defined. Studies have indicated that transmembrane helix 7 (TMH7) and the highly conserved NPXXY motif can be subject to different conformational changes in response to biased ligands and could therefore participate in a molecular mechanism to trigger β-arrestin recruitment. " Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 "The cannabinoid CB1 receptor is the most abundant G protein coupled receptor (GPCR) in the central nervous system, which mediates the functional response to endocannabinoids and Cannabis compounds. A variety of ligands for CB1 receptors have been developed as promising drug candidates for the treatment of neurological disorders. New high-resolution structures of CB1 receptor in different functional states have significantly improved our molecular understanding of CB1 ligand interactions, selectivity, receptor activation and allosteric modulation. These advances have paved the way for development of novel ligands for different therapeutic applications. In this review, we describe the structural determinants for modulation of CB1 receptors by different types of ligands, as well as the differences between CB1 and its homologous, the CB2 receptor. LINKED ARTICLES: This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). " Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 Gαs and Gαq/11 protein coupling bias of two AVPR2 mutants (R68W and V162A) that cause nephrogenic diabetes insipidus "Loss-of-function mutations of the arginine vasopressin receptor 2 gene (AVPR2) cause Nephrogenic diabetes insipidus (NDI). AVPR2 is a kind of G protein coupled receptor (GPCR) and mainly couples with Gαs protein leading to cAMP accumulation in the cell as a secondary messenger. Recent studies showed that some AVPR2 mutations could cause biased Gαq/11 protein coupling rather than Gαs. Investigation into the characterization of biased receptors may give insights into the relationship between the conformational change of the receptor because of the mutation and related downstream signaling. In this study, R68W and V162A were analyzed to whether they show a bias to Gαs or Gαq/11 proteins. Their functionality in terms of cAMP production via Gαs protein coupling was decreased compared to the wild-type receptor. On the other hand, they showed the ability to couple with Gαq/11 protein and make Ca2+ mobilization at different levels in the cell. R68W showed bias to coupling with Gαq/11 protein rather than V162A and wild-type receptor. Studies about the Gα protein coupling bias of mutant AVPR2s may broaden our understanding of the relationship between the changed conformation of the receptor and consequently activated signaling pathways, and also may shed light on the development of more effective new therapeutics." Read more at the source #DrGPCR #GPCR #IndustryNews

August 2022 Production of human A 2A AR in lipid nanodiscs for 19 F-NMR and single-molecule fluorescence spectroscopy "We describe production of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR) for 19F-NMR and single-molecule fluorescence (SMF) spectroscopy. We explain in detail steps shared between the two sample preparation strategies, including expression and isolation of A2AAR and assembly of A2AAR in lipid nanodiscs and procedures for incorporation of either 19F-NMR or fluorescence probes. Protocols for SMF experiments include sample setup, data acquisition, data processing, and error analysis. For complete details on the use and execution of this protocol, please refer to Wei et al. (2022) and Sušac et al. (2018)." Read more at the source #DrGPCR #GPCR #IndustryNews