Class Of G-protein Coupled Receptors Research Articles

Structural and functional analysis of Ccr1l1, a Rodentia-restricted eosinophil-selective chemokine receptor homologue

Mouse Ccr1l1 (Ccr1-like 1) encodes an orphan G-protein-coupled receptor (GPCR) with the highest homology to the inflammatory and highly promiscuous chemokine receptors Ccr1 and Ccr3 (70 and 50% amino acid identity, respectively). Ccr1l1 was first cloned in 1995, yet current knowledge of this putative chemokine receptor is limited to its gene organization and chromosomal localization. Here we report that Ccr1l1 is a Rodentia-specific gene selectively expressed in eosinophils. However, eosinophil phenotypes, development, and responsiveness to chemokines were all normal in naïve Ccr1l1 knockout mice. We demonstrate for the first time that recombinant Ccr1l1 is expressed on the plasma membrane of transfected cells and contains an extracellular N terminus and an intracellular C terminus, consistent with GPCR topology. Using receptor internalization, β-arrestin recruitment, calcium flux, and chemotaxis assays, we excluded all 37 available mouse chemokines, including Ccr1 ligands, and two viral chemokines as Ccr1l1 ligands, and demonstrated that mouse Ccr1, but not Ccr1l1, exhibits constitutive signaling activity. However, sequence analysis and structural modeling revealed that Ccr1l1 is well equipped to act as a classical signaling GPCR, with N-terminal sulfotyrosines as the only signaling and chemokine-binding determinant absent in Ccr1l1. Hereof, we show that a sulfatable N-terminal Ccr1 Y18 residue is essential for chemotaxis and calcium responses induced by Ccl3 and Ccl9/10, but substituting the corresponding Ccr1l1 F19 residue with tyrosine failed to confer responsiveness to Ccr1 ligands. Although Ccr1l1 remains an extreme outlier in the chemokine receptor family, our study supports that it might respond to unidentified mouse chemokine ligands in eosinophil-driven immune responses.

A synthetic method to assay adhesion-family G-protein coupled receptors. Determination of the G-protein coupling profile of ADGRG6(GPR126)

G-protein coupled receptors (GPCRs) are the largest family of membrane-spanning receptors in metazoans and mediate diverse biological processes such as chemotaxis, vision, and neurotransmission. Adhesion GPCRs represent an understudied class of GPCRs. Adhesion GPCRs (ADGRs) are activated by an intrinsic proteolytic mechanism executed by the G-protein autoproteolysis inducing domain that defines this class of GPCRs. It is hypothesized that agonist ligands modulate the proteolyzed receptor to regulate the activity of a tethered agonist peptide that is an intramolecular activator of ADGRs. The mechanism of activation of ADGRs in physiological settings is unclear and the toolbox for interrogating ADGR physiology in cellular models is limited. Therefore, we generated a novel enterokinase-activated tethered ligand system for ADGRG6(GPR126). Enterokinase addition to cells expressing a synthetic ADGRG6 protein induced potent and efficacious signal transduction through heterotrimeric G-protein coupled second messenger pathways including cyclic nucleotide production, intracellular calcium mobilization, and GPCR-pathway linked reporter gene induction. These studies support the hypothesis that ADGRG6(GPR126) is coupled to multiple heterotrimeric G-proteins: including Gαs, Gαq, and Gα12. This novel assay method is robust, specific, and compatible with numerous cell pharmacology approaches. We present a new tool for determination of the biological function of ADGRs and the identification of ligands that engage these receptors.

Insights into the Activation Mechanism of the ALX/FPR2 Receptor.

The formyl peptide receptor 2 (ALX/FPR2), a G-protein-coupled receptor (GPCR), plays an important role in host defense and inflammation. This receptor can be driven as pro- or anti-inflammatory depending on its agonist, such as N-formyl-Met-Leu-Phe-Lys (fMLFK) and resolvin D1 (RvD1) or its aspirin-triggered 17 (R)-epimer, AT-RvD1, respectively. However, the activation mechanism of ALX/FPR2 by pro- and anti-inflammatory agonists remains unclear. In this work, on the basis of molecular dynamics simulations, we evaluated a model of the ALX/FPR2 receptor activation process using two agonists, fMLFK and AT-RvD1, with opposite effects. The simulations by both fMLFK and AT-RvD1 induced the ALX/FPR2 activation through a set of receptor-core residues, in particular, R205, Q258, and W254. In addition, the activation was dependent on the disruption of electrostatic interactions in the cytoplasmic region of the receptor. We also found that in the AT-RvD1 simulations, the position of the H8 helix was similar to that of the same helix in other class-A GPCRs coupled to arrestin. Thus our results shed light on the mechanism of activation of the ALX/FPR2 receptor by pro-inflammatory and pro-resolution agonists.

Aspergillus fumigatus G-Protein Coupled Receptors GprM and GprJ Are Important for the Regulation of the Cell Wall Integrity Pathway, Secondary Metabolite Production, and Virulence.

G-protein coupled receptors (GPCRs) are extracellular signaling receptors that sense environmental cues. Fungi sense their environment primarily through GPCR-mediated signaling pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. Aspergillus fumigatus is an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that present a wide range of clinical manifestations. Here, we investigate in detail the role of the GPCRs GprM and GprJ in growth and gene expression. GprM and GprJ are important for melanin production and the regulation of the cell wall integrity (CWI) pathway. Overexpression of gprM and gprJ causes a 20 and 50% reduction in growth rate compared to the wild-type (WT) strain and increases sensitivity to cell wall-damaging agents. Phosphorylation of the CWI protein kinase MpkA is increased in the ΔgprM and ΔgprJ strains and decreased in the overexpression mutants compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. Transcriptome sequencing suggests that GprM and GprJ negatively regulate genes encoding secondary metabolites (SMs). Mass spectrometry analysis confirmed that the production of fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, and fumitremorgin is reduced in the ΔgprM and ΔgprJ strains, at least partially through the activation of MpkA. Overexpression of grpM also resulted in the regulation of many transcription factors, with AsgA predicted to function downstream of GprM and MpkA signaling. Finally, we show that the ΔgprM and ΔgprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis.IMPORTANCEA. fumigatus is the main etiological agent of invasive pulmonary aspergillosis, a life-threatening fungal disease that occurs in severely immunocompromised humans. Withstanding the host environment is essential for A. fumigatus virulence, and sensing of extracellular cues occurs primarily through G-protein coupled receptors (GPCRs) that activate signal transduction pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. The A. fumigatus genome encodes 15 putative classical GPCRs, with only three having been functionally characterized to date. In this work, we show that the two GPCRs GprM and GprJ regulate the phosphorylation of the mitogen-activated protein kinase MpkA and thus control the regulation of the cell wall integrity pathway. GprM and GprJ are also involved in the regulation of the production of the secondary metabolites fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, melanin, and fumitremorgin, and this regulation partially occurs through the activation of MpkA. Furthermore, GprM and GprJ are important for virulence in the insect model Galleria mellonella This work therefore functionally characterizes two GPCRs and shows how they regulate several intracellular pathways that have been shown to be crucial for A. fumigatus virulence.

An investigation into the allosteric mechanism of GPCR A2A adenosine receptor with trajectory-based information theory and complex network model

G protein-coupled receptors (GPCRs), a large superfamily of transmembrane (TM) proteins, allosterically transduce the signal of ligand binding in the extracellular (EC) domain to couple to effector proteins in the intracellular (IC) domain, therefore forming the largest class of drug targets. The A2A adenosine receptor (A2AAR), a class-A GPCR, has been extensively studied as it offers numerous possibilities for therapeutic applications. However, the mechanism of allosteric communication between EC and IC domains is not completely clear. In this work, we utilize torsional mutual information to quantify the correlated motions of residue pairs from its molecular dynamics (MD) simulation trajectories, and further use the complex network model to obtain allosteric pipelines and hubs. The identified allosteric communication pipelines mainly transmit the signal from EC domain to the cytoplasmic ends of TM helix 5 (TM5), TM6 and TM7. The allosteric hubs, mostly located at TM5, TM6 and TM7, play an important role in mediating allosteric signal transmission to keep the receptor rigid and prevent G protein from binding to IC domain, which can explain the reason why their mutations distant from ligand-binding site do not affect the ligand binding affinity but affect the ligand efficacy. Additionally, we identify the key residues located in antagonist ZM241385 binding pocket which mediate multiple allosteric pathways and have been experimentally proven to play a critical role in affecting the ligand potency. This study is helpful for understanding the allosteric communication mechanism of A2AAR, and can provide valuable information for the structure-based drug design of GPCRs. Communicated by Ramaswamy H. Sarma

From Venom to AChE Inhibitor: Design, Molecular Modeling, and Synthesis of a Peptidic Inhibitor of AChE

Alzheimer’s disease (AD) is an irreversible and progressive brain disorder that slowly destroys memory and cognitive skills. The current treatment of AD mainly focused on the restoring of ACh levels through acetylcholinesterase (AChE) inhibition. Peptides are a unique class of pharmaceutical compounds that have privilege over small molecules, especially in the realm of protein–protein interactions and G protein-coupled receptor (GPCR) inhibitors. We applied a rational structure-based virtual design approach to discover new peptidic inhibitors of AChE. In this regard, conformational space in the fasciculin II (Fas) and AChE complex was evaluated utilizing MD simulation, principal component analysis and clustering to figure out possible interactions of Fas and AChE. Assessment of Fas–AChE interactions by visual evaluation and alanine scanning led to the design of 10 peptides. The highest scored peptide (p2) was selected and synthesized using SPPS. Based on Ellman's test, the inhibitory activity of p2 against AChE was 51.2 ± 8.1 µM. The kinetics study of the enzyme inhibition in accompany with molecular modeling results revealed that p2 was a mixed-type reversible inhibitor of AChE. The DNRMLRTTRY peptide was considerable inhibitor of AChE. Peptides have the merit of being big enough to inhibit PPI and GPCR class B with a wide binding site. But possible peptidic chemical space is too large to be evaluated by the classical peptide synthesis methods. In the present contribution, we introduced a rational in silico peptide design approach that led to the considerable peptidic inhibitor of AChE.

Virtual Screening of Human Class-A GPCRs Using Ligand Profiles Built on Multiple Ligand–Receptor Interactions

G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins responsible for cellular signal transductions. Identification of therapeutic compounds to regulate physiological processes is an important first step of drug discovery. We proposed MAGELLAN, a novel hierarchical virtual-screening (VS) pipeline, which starts with low-resolution protein structure prediction and structure-based binding-site identification, followed by homologous GPCR detections through structure and orthosteric binding-site comparisons. Ligand profiles constructed from the homologous ligand–GPCR complexes are then used to thread through compound databases for VS. The pipeline was first tested in a large-scale retrospective screening experiment against 224 human Class A GPCRs, where MAGELLAN achieved a median enrichment factor (EF) of 14.38, significantly higher than that using individual ligand profiles. Next, MAGELLAN was examined on 5 and 20 GPCRs from two public VS databases (DUD-E and GPCR-Bench) and resulted in an average EF of 9.75 and 13.70, respectively, which compare favorably with other state-of-the-art docking- and ligand-based methods, including AutoDock Vina (with EF = 1.48/3.16 in DUD-E and GPCR-Bench), DOCK 6 (2.12/3.47 in DUD-E and GPCR-Bench), PoLi (2.2 in DUD-E), and FINDSITECcomb2.0 (2.90 in DUD-E). Detailed data analyses show that the major advantage of MAGELLAN is attributed to the power of ligand profiling, which integrates complementary methods for ligand–GPCR interaction recognition and thus significantly improves the coverage and sensitivity of VS models. Finally, cases studies on opioid and motilin receptors show that new connections between functionally related GPCRs can be visualized in the minimum spanning tree built on the similarities of predicted ligand-binding ensembles, suggesting a novel use of MAGELLAN for GPCR deorphanization.

FLIM–FRET-Based Structural Characterization of a Class-A GPCR Dimer in the Cell Membrane

Class-A G protein-coupled receptors (GPCRs) are known to homo-dimerize in the membrane. Yet, methods to characterize the structure of GPCR dimer in the native environment are lacking. Accordingly, the molecular basis and functional relevance of the class-A GPCR dimerization remain unclear. Here, we present the dimeric structural model of GPR17 in the cell membrane. The dimer mainly involves transmembrane helix 5 (TM5) at the interface, with F229 in TM5, a critical residue. An F229A mutation makes GPR17 monomeric regardless of the expression level of the receptor. Monomeric mutants of GPR17 display impaired ERK1/2 activation and cannot be properly internalized upon agonist treatment. Conversely, the F229C mutant is cross-linked as a dimer and behaves like wild-type. Importantly, the GPR17 dimer structure has been modeled using sparse inter-protomer FRET distance restraints obtained from fluorescence lifetime imaging microscopy. The same approach can be applied to characterizing the interactions of other important membrane proteins in the cell.

1705-P: Adipocyte Specific Ablation of P2Y14R Improves Glucose Metabolism in Mice with Diet-Induced Obesity

Adipose tissue is one of the major tissues in body regulating glucose and energy homeostasis. White adipose tissue (iWAT) serves as primary site of energy storage while brown (BAT) and beige AT play a major role in energy expenditure. AT also exerts endocrine effects by secreting various adipokines/batokines regulating whole body glucose metabolism. Diverse G protein-coupled receptors (GPCRs) are expressed in adipocytes and thus have emerged as potential targets for novel antidiabetic drugs. P2Y purinergic receptors are a class of GPCRs activated by nucleotides and nucleotide sugars. The purinergic P2Y14 receptor (P2Y14R) is natively activated by UDP-glucose. P2Y14R is expressed in white as well as brown adipocytes, and receptor mRNA levels was increased in iWAT due to high fat feeding. The potential role of P2Y14R in AT with respect to maintaining whole body glucose homeostasis remains unexplored. To this end, we generated a knockout mouse model lacking P2Y14R selectively in adipocytes (Ad-P2Y14-KO) using Cre/loxP technology. Interestingly, Ad-P2Y14-KO consuming a high fat diet gained less weight than the control mice. Body composition revealed that the decrease in body weight gain in Ad-P2Y14-KO was due to a decrease in fat mass. Decreased body weight resulted in improved glucose tolerance and insulin sensitivity in Ad-P2Y14-KO. Moreover, fasting blood glucose and fed plasma insulin levels were decreased in Ad-P2Y14-KO. Circulating levels of adipokines such as leptin were decreased in knockout mice, indicating increased sensitivity to leptin in Ad-P2Y14-KO. Interestingly, a lack of P2Y14R signaling increased lipolysis in adipocytes, as plasma free fatty acids levels was elevated in Ad-P2Y14-KO in the fasting state. Liver weight was decreased in Ad-P2Y14-KO indicating a lack of ectopic deposition of fat due to enhanced lipolysis. Mechanistic studies are underway to link the observed phenotype with P2Y14R signaling in adipocytes. Disclosure S. Jain: None. K.A. Jacobson: None.

New Insights from IGF-IR Stimulating Activity Analyses: Pathological Considerations.

Insulin-like growth factor-I (IGF-I) and insulin-like growth factor-II (IGF-II) play a crucial factor in the growth, differentiation and survival of cells in health and disease. IGF-I and IGF-II primarily activate the IGF-I receptor (IGF-IR), which is present on the cell surface. Activation of the IGF-IR stimulates multiple pathways which finally results in multiple biological effects in a variety of tissues and cells. In addition, activation of the IGF-IR has been found to be essential for the growth of cancers. The conventional view in the past was that the IGF-IR was exclusively a tyrosine kinase receptor and that phosphorylation of tyrosine residues, after binding of IGF-I to the IGF-IR, started a cascade of post-receptor events. Recent research has shown that this view was too simplistic. It has been found that the IGF-IR also has kinase-independent functions and may even emit signals in the unoccupied state through some yet-to-be-defined non-canonical pathways. The IGF-IR may further form hybrids with the insulin receptors but also with receptor tyrosine kinases (RTKs) outside the insulin-IGF system. In addition, the IGF-IR has extensive cross-talk with many other receptor tyrosine kinases and their downstream effectors. Moreover, there is now emerging evidence that the IGF-IR utilizes parts of the G-protein coupled receptor (GPCR) pathways: the IGF-IR can be considered as a functional RTK/GPCR hybrid, which integrates the kinase signaling with some IGF-IR mediated canonical GPCR characteristics. Like the classical GPCRs the IGF-IR can also show homologous and heterologous desensitization. Recently, it has been found that after activation by a ligand, the IGF-IR may be translocated into the nucleus and function as a transcriptional cofactor. Thus, in recent years, it has become clear that the IGF-IR signaling pathways are much more complex than first thought. Therefore a big challenge for the (near) future will be how all the new knowledge about IGF-IR signaling can be translated into the clinical practice and improve diagnosis and treatment of diseases.

Comparative transcriptomics supports the presence of G protein‐coupled receptor‐based signaling in unicellular marine eukaryotes

AbstractBeing abundant primary producers, single‐celled eukaryotic diatoms and dinoflagellates dominate marine food webs and significantly impact the ecology of the oceans. These organisms face competition and predation which have contributed to the evolution of chemical defenses and their ability to sense chemical cues. Elucidating how these chemical cues are perceived by organisms requires identification of proteins involved in relaying chemical information to cells. The functions of G protein‐coupled receptor (GPCR) signaling diversified over evolutionary time in multicellular organisms, but the role that GPCR signaling plays in unicellular eukaryotes remains largely unknown. Using comparative transcriptomic analysis, we identified repertoires of GPCR signaling proteins from 81 diatom and 36 dinoflagellate transcriptomes. Diatoms and dinoflagellates express putative GPCRs that are highly diverged from known metazoan GPCRs along with well‐conserved core components of GPCR signaling pathways. Unlike other eukaryotes that express multiple classes of GPCRs, diatoms and dinoflagellates were each found to express a single, major class of putative GPCRs with greatest similarity to class C and rhodopsin‐like GPCRs, respectively. Furthermore, only 4.2% of the GPCR‐like transcripts from dinoflagellates were predicted to be true GPCRs compared to 48% in diatoms. These results suggest that further expansion of GPCRs did not take place within each of these two major phytoplankton groups, and that dinoflagellate GPCRs are more divergent from known GPCRs than are diatom GPCRs. Our results provide additional evidence for the global existence of relatively ancestral GPCR signaling machinery in marine phytoplankton.

Extramembranous Regions in G Protein-Coupled Receptors: Cinderella in Receptor Biology?

G protein-coupled receptors (GPCRs) are the largest class of membrane proteins involved in signal transduction and are characterized by seven transmembrane domain architecture interconnected by extra- and intracellular loops. These loops, along with the N- and C-terminal domains, constitute the extramembranous regions in GPCRs. These regions, accounting for ~ 40% or more amino acid residues across different GPCR classes, are distinct from the conserved transmembrane domains in terms of nonconservation of sequence, diversity in length, and conformational heterogeneity. Due to technical challenges in exploring the molecular basis underlying the relation between structure, dynamics, and function in these regions, their contribution to GPCR organization and signaling remain underappreciated. Despite existingliterature on the involvement of GPCR loops in numerous aspects of GPCR biology, the functional relevance of GPCR loops in the context of their inherent conformational heterogeneity and probable membrane interaction are not well understood. This review focuses on highlighting these aspects of GPCR extramembranous regions in the overall context of GPCR organization, dynamics, and biology. We envision that a judicious combination of insights obtained from structured transmembrane domains and disordered extramembranous regions in GPCRs would be crucial in arriving at a comprehensive understanding of GPCR structure, function, and dynamics, thereby leading to efficient drug discovery.

Cryo-Electron Microscopy: Moving Beyond X-Ray Crystal Structures for Drug Receptors and Drug Development.

Electron cryo-microscopy (cryo-EM) has revolutionized structure determination of membrane proteins and holds great potential for structure-based drug discovery. Here we discuss the potential of cryo-EM in the rational design of therapeutics for membrane proteins compared to X-ray crystallography. We also detail recent progress in the field of drug receptors, focusing on cryo-EM of two protein families with established therapeutic value, the γ-aminobutyric acid A receptors (GABAARs) and G protein-coupled receptors (GPCRs). GABAARs are pentameric ion channels, and cryo-EM structures of physiological heteromeric receptors in a lipid environment have uncovered the molecular basis of receptor modulation by drugs such as diazepam. The structures of ten GPCR-G protein complexes from three different classes of GPCRs have now been determined by cryo-EM. These structures give detailed insights into molecular interactions with drugs, GPCR-G protein selectivity, how accessory membrane proteins alter receptor-ligand pharmacology, and the mechanism by which HIV uses GPCRs to enter host cells.

A Machine Learning Approach for the Discovery of Ligand-Specific Functional Mechanisms of GPCRs.

G protein-coupled receptors (GPCRs) play a key role in many cellular signaling mechanisms, and must select among multiple coupling possibilities in a ligand-specific manner in order to carry out a myriad of functions in diverse cellular contexts. Much has been learned about the molecular mechanisms of ligand-GPCR complexes from Molecular Dynamics (MD) simulations. However, to explore ligand-specific differences in the response of a GPCR to diverse ligands, as is required to understand ligand bias and functional selectivity, necessitates creating very large amounts of data from the needed large-scale simulations. This becomes a Big Data problem for the high dimensionality analysis of the accumulated trajectories. Here we describe a new machine learning (ML) approach to the problem that is based on transforming the analysis of GPCR function-related, ligand-specific differences encoded in the MD simulation trajectories into a representation recognizable by state-of-the-art deep learning object recognition technology. We illustrate this method by applying it to recognize the pharmacological classification of ligands bound to the 5-HT2A and D2 subtypes of class-A GPCRs from the serotonin and dopamine families. The ML-based approach is shown to perform the classification task with high accuracy, and we identify the molecular determinants of the classifications in the context of GPCR structure and function. This study builds a framework for the efficient computational analysis of MD Big Data collected for the purpose of understanding ligand-specific GPCR activity.

A predictive computational model reveals that GIV/girdin serves as a tunable valve for EGFR-stimulated cyclic AMP signals.

Cellular levels of the versatile second messenger cyclic (c)AMP are regulated by the antagonistic actions of the canonical G protein → adenylyl cyclase pathway that is initiated by G-protein–coupled receptors (GPCRs) and attenuated by phosphodiesterases (PDEs). Dysregulated cAMP signaling drives many diseases; for example, its low levels facilitate numerous sinister properties of cancer cells. Recently, an alternative paradigm for cAMP signaling has emerged in which growth factor–receptor tyrosine kinases (RTKs; e.g., EGFR) access and modulate G proteins via a cytosolic guanine-nucleotide exchange modulator (GEM), GIV/girdin; dysregulation of this pathway is frequently encountered in cancers. In this study, we present a network-based compartmental model for the paradigm of GEM-facilitated cross-talk between RTKs and G proteins and how that impacts cellular cAMP. Our model predicts that cross-talk between GIV, Gαs, and Gαi proteins dampens ligand-stimulated cAMP dynamics. This prediction was experimentally verified by measuring cAMP levels in cells under different conditions. We further predict that the direct proportionality of cAMP concentration as a function of receptor number and the inverse proportionality of cAMP concentration as a function of PDE concentration are both altered by GIV levels. Taking these results together, our model reveals that GIV acts as a tunable control valve that regulates cAMP flux after growth factor stimulation. For a given stimulus, when GIV levels are high, cAMP levels are low, and vice versa. In doing so, GIV modulates cAMP via mechanisms distinct from the two most often targeted classes of cAMP modulators, GPCRs and PDEs.

Conserved 2nd Residue of Helix 8 of GPCR May Confer the Subclass-Characteristic and Distinct Roles through a Rapid Initial Interaction with Specific G Proteins.

To obtain a systematic view of the helix-8-second residue responsible for G protein-coupled receptor (GPCR)–G protein initial specific interactions, 786 human GPCRs were subclassified based on the pairs of agonist groups and target G proteins and compared with their conserved second residue of helix 8. Of 314 non-olfactory and deorphanized GPCRs, 273 (87%) conserved single amino acids in the subclasses, while 93 (58%) of the 160 subclasses possessed only a single GPCR member. Class B, C, Frizzled, and trace amine-associated GPCRs demonstrated 100% conservation, whereas class I and II olfactory and vomeronasal 1 receptors demonstrated much lower rates of conservation (20–47%). These conserved residues are characteristic of GPCR classes and G protein subtypes and confer their functionally-distinct roles.

Skeletal Muscle-Specific Activation of Gq Signaling Maintains Glucose Homeostasis.

Skeletal muscle (SKM) insulin resistance plays a central role in the pathogenesis of type 2 diabetes. Because G-protein-coupled receptors (GPCRs) represent excellent drug targets, we hypothesized that activation of specific functional classes of SKM GPCRs might lead to improved glucose homeostasis in type 2 diabetes. At present, little is known about the in vivo metabolic roles of the various distinct GPCR signaling pathways operative in SKM. In this study, we tested the hypothesis that selective activation of SKM Gq signaling can improve SKM glucose uptake and whole-body glucose homeostasis under physiological and pathophysiological conditions. Studies with transgenic mice expressing a Gq-linked designer GPCR selectively in SKM cells demonstrated that receptor-mediated activation of SKM Gq signaling greatly promoted glucose uptake into SKM and significantly improved glucose homeostasis in obese, glucose-intolerant mice. These beneficial metabolic effects required the activity of SKM AMPK. In contrast, obese mutant mice that lacked both Gαq and Gα11 selectively in SKM showed severe deficits in glucose homeostasis. Moreover, GPCR-mediated activation of Gq signaling also stimulated glucose uptake in primary human SKM cells. Taken together, these findings strongly suggest that agents capable of enhancing SKM Gq signaling may prove useful as novel antidiabetic drugs.

Biased Signaling and Allosteric Modulation at the FSHR.

Knowledge on G protein-coupled receptor (GPCRs) structure and mechanism of activation has profoundly evolved over the past years. The way drugs targeting this family of receptors are discovered and used has also changed. Ligands appear to bind a growing number of GPCRs in a competitive or allosteric manner to elicit balanced signaling or biased signaling (i.e., differential efficacy in activating or inhibiting selective signaling pathway(s) compared to the reference ligand). These novel concepts and developments transform our understanding of the follicle-stimulating hormone (FSH) receptor (FSHR) biology and the way it could be pharmacologically modulated in the future. The FSHR is expressed in somatic cells of the gonads and plays a major role in reproduction. When compared to classical GPCRs, the FSHR exhibits intrinsic peculiarities, such as a very large NH2-terminal extracellular domain that binds a naturally heterogeneous, large heterodimeric glycoprotein, namely FSH. Once activated, the FSHR couples to Gαs and, in some instances, to other Gα subunits. G protein-coupled receptor kinases and β-arrestins are also recruited to this receptor and account for its desensitization, trafficking, and intracellular signaling. Different classes of pharmacological tools capable of biasing FSHR signaling have been reported and open promising prospects both in basic research and for therapeutic applications. Here we provide an updated review of the most salient peculiarities of FSHR signaling and its selective modulation.

Involvement of the Adhesion GPCRs Latrophilins in the Regulation of Insulin Release

Insulin secretion from pancreatic β cells is a highly complex and tightly regulated process. Its dysregulation is one characteristic of type 2 diabetes, and thus, an in-depth understanding of the mechanisms controlling insulin secretion is essential for rational therapeutic intervention. G-protein-coupled receptors (GPCRs) have been established as major regulators of insulin exocytosis. Recent studies also suggest the involvement of adhesion GPCRs, a non-prototypical class of GPCRs. Here, we identify latrophilins, which belong to the class of adhesion GPCRs, to be highly expressed in different cell types of pancreatic islets. Invitro and exvivo analyses show that distinct splice variants of the latrophilin LPHN3/ADGRL3 decrease insulin secretion from pancreatic β cells by reducing intracellular cyclic AMP levels via the Gi-mediated pathway. Our data highlight the key role of LPHN3 in modulating insulin secretion and its potential as therapeutic target for type 2 diabetes.

Mechanisms of Pancreatic Injury Induced by Basic Amino Acids Differ Between L-Arginine, L-Ornithine, and L-Histidine.

Pancreatic acinar cells require high rates of amino acid uptake for digestive enzyme synthesis, but excessive concentrations can trigger acute pancreatitis (AP) by mechanisms that are not well understood. We have used three basic natural amino acids L-arginine, L-ornithine, and L-histidine to determine mechanisms of amino acid-induced pancreatic injury and whether these are common to all three amino acids. Caffeine markedly inhibited necrotic cell death pathway activation in isolated pancreatic acinar cells induced by L-arginine, but not L-ornithine, whereas caffeine accelerated L-histidine-induced cell death. Both necroptosis inhibitors of RIPK1 and RIPK3 and a necroptosis activator/apoptosis inhibitor z-VAD increased cell death caused by L-histidine, but not L-arginine or L-ornithine. Cyclophilin D knock-out (Ppif-/-) significantly attenuated cell death induced by L-histidine, but not L-arginine, or L-ornithine. Allosteric modulators of calcium-sensing receptor (CaSR) and G-protein coupled receptor class C group 6 member A (GPRC6A) had inhibitory effects on cell death induced by L-arginine but not L-ornithine or L-histidine. We developed a novel amino acid-induced AP murine model with high doses of L-histidine and confirmed AP severity was significantly reduced in Ppif-/- vs. wild type mice. In L-arginine-induced AP neither Ppif-/-, caffeine, or allosteric modulators of CaSR or GPRC6A reduced pancreatic damage, even though CaSR inhibition with NPS-2143 significantly reduced pancreatic and systemic injury in caerulein-induced AP. These findings demonstrate marked differences in the mechanisms of pancreatic injury induced by different basic amino acids and suggest the lack of effect of treatments on L-arginine-induced AP may be due to conversion to L-ornithine in the urea cycle.