G-protein Coupled Receptor Member Research Articles

GPR160 regulates the self-renewal and pluripotency of mouse embryonic stem cells via JAK1/STAT3 signal pathway

G-protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors and regulate various physiological and pathological processes. Despite extensive studies, the roles of GPCRs in mouse embryonic stem cells (mESCs) represent a significant data gap. Here, we show that GPR160, a class A member of GPCRs, is dramatically downregulated concurrent with mESC differentiation into embryoid bodies in vitro. Knockdown of GPR160 leads to downregulation of the expression of pluripotency-associated transcription factors and upregulation of the expression of lineage markers, accompanying with the arrest of the mESC cell-cycle in the G0/G1 phase. RNA-seq analysis shows that GPR160 participates in the JAK/STAT signaling pathway crucial for maintaining ESC stemness, and the knockdown of GPR160 results in the downregulation of STAT3 phosphorylation level, which in turn is partially rescued by colivelin, a STAT3 activator. Constant with these observations, GPR160 physically interacts with JAK1, and cooperates with leukemia inhibitory factor receptor (LIFR) and gp130 to activate the STAT3 pathway. In summary, our results suggest that GPR160 regulates mESC self-renewal and pluripotency by interacting with the JAK1-LIFR-gp130 complex to mediate the JAK1/STAT3 signaling pathway.

Segregation of nascent GPCRs in the ER-to-Golgi transport by CCHCR1 via direct interaction.

G protein-coupled receptors (GPCRs) constitute the largest superfamily of cell surface signaling proteins that share a common structural topology. When compared with agonist-induced internalization, how GPCRs are sorted and delivered to functional destinations after synthesis in the endoplasmic reticulum (ER) is much less well understood. Here, we demonstrate that depletion of coiled-coil α-helical rod protein 1 (CCHCR1) by siRNA and CRISPR-Cas9 significantly inhibits surface expression and signaling of α2A-adrenergic receptor (α2A-AR; also known as ADRA2A), without affecting α2B-AR. Further studies show that CCHCR1 depletion specifically impedes α2A-AR export from the ER to the Golgi, but not from the Golgi to the surface. We also demonstrate that CCHCR1 selectively interacts with α2A-AR. The interaction is mediated through multiple domains of both proteins and is ionic in nature. Moreover, mutating CCHCR1-binding motifs significantly attenuates ER-to-Golgi export, surface expression and signaling of α2A-AR. Collectively, these data reveal a novel function for CCHCR1 in intracellular protein trafficking, indicate that closely related GPCRs can be sorted into distinct ER-to-Golgi transport routes by CCHCR1 via direct interaction, and provide important insights into segregation and anterograde delivery of nascent GPCR members.

Recent Studies on Serotonin 5-HT2A Receptor Antagonists in Medicinal Chemistry: A Last Decades Survey.

In the last decades, much attention has been paid to the functioning of receptors to understand better how they work with various chemical motifs. Among different families, G-proteincoupled receptor (GPCR) families have drawn much attention in the twenty-first century. They are the most prominent signal transducer across the cell membrane, comprising thousand-odd proteins. One of the members of GPCRs is the serotonin 2A (5-HT2A) receptor, which has been associated with complex etiological mental illnesses. In this survey, we collected data on 5-HT2A, i.e., the role of 5- HT2A receptors in human and animal analogy, various binding site functionalities, advanced effects, and synthetic aspects.

Insight into Evolution and Conservation Patterns of B1-Subfamily Members of GPCR.

The diverse, evolutionary architectures of proteins can be regarded as molecular fossils, tracing a historical path that marks important milestones across life. The B1-subfamily of GPCRs (G-protein-coupled receptors) are medically significant proteins that comprise 15 transmembrane receptor proteins in Homo sapiens. These proteins control the intracellular concentration of cyclic AMP as well as various vital processes in the body. However, little is known about the evolutionary correlation and conservational blueprint of this GPCR subfamily. We performed a comprehensive analysis to understand the evolutionary architecture among 13 members of the B1-subfamily. Multiple sequence alignment analysis exhibited six multiple sequence aligned blocks and five highly aligned blocks. Molecular phylogenetics indicated that CRHR1 and CRHR2 share a typical ancestral relationship and are siblings in 100% bootstrap replications with a total of 24 nodes observed in the cladogram. CRHR2 has the maximum number of extremely conserved amino acids followed by ADCYAP1R1. The longest continuous number sequence logos (74) were found between sequence location 349 and 423, and consequently, the maximum and minimum logo height recorded was 3.6 bits and 0.18 bits, respectively. Finally, to understand the model and pattern of evolutionary relatedness, the conservation blueprint, and the diversification among the members of a protein family, GPCR distribution from several species throughout the animal kingdom was analysed. Together, the study provides an evolutionary insight and offers a rapid method to explore the potential of depicting the evolutionary relationship, conservation blueprint, and diversification among the B1-subfamily of GPCRs using bioinformatics, algorithm analysis, and mathematical models.Electronic supplementary materialThe online version of this article (10.1007/s10989-020-10043-5) contains supplementary material, which is available to authorized users.

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.

Specific TBC Domain‐Containing Proteins Control the ER‐Golgi‐Plasma Membrane Trafficking of GPCRs

G protein‐coupled receptors (GPCRs) constitute the largest superfamily of cell‐surface signaling proteins. However, the molecular mechanisms underlying their cell surface delivery after synthesis remain poorly understood. Here we screen the TBC domain‐containing proteins, putative Rab GTPase‐activating proteins (GAPs), in the intracellular trafficking of GPCRs and identify several TBC proteins that activity‐dependently regulate the anterograde transport, en route from the endoplasmic reticulum to the Golgi or from the Golgi to the cell surface, of several prototypic GPCR members without affecting other plasma membrane proteins. We also show that TBC1D6 functions as a GAP for Rab26, physically associates with Rab26, and attenuates Rab26 interaction with GPCRs. Furthermore, both overexpression and depletion of TBC1D6 inhibit the post‐Golgi traffic of GPCRs. These data demonstrate for the first time the important roles of the TBC proteins in forward trafficking of nascent GPCRs and reveal novel regulatory mechanisms of GPCR targeting to the functional destination.Support or Funding InformationNIH R01GM118915This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Deregulation of Frizzled Receptors in Hepatocellular Carcinoma.

G protein-coupled receptors (GPCRs) have a substantial role in tumorigenesis and are described as a “cancer driver”. Aberrant expression or activation of GPCRs leads to the deregulation of downstream signaling pathways, thereby promoting cancer progression. In hepatocellular carcinoma (HCC), the Wnt signaling pathway is frequently activated and it is associated with an aggressive HCC phenotype. Frizzled (FZD) receptors, a family member of GPCRs, are known to mediate Wnt signaling. Accumulating findings have revealed the deregulation of FZD receptors in HCC and their functional roles have been implicated in HCC progression. Given the important role of FZD receptors in HCC, we summarize here the expression pattern of FZD receptors in HCC and their corresponding functional roles during HCC progression. We also further review and highlight the potential targeting of FZD receptors as an alternative therapeutic strategy in HCC.

Beta-Arrestin 1 Mediates Liver Thyrotropin Regulation of Cholesterol Conversion Metabolism via the Akt-Dependent Pathway.

After activation, G protein-coupled receptors (GPCRs) are desensitized by β-arrestins (ARRBs). Moreover, ARRBs can initiate a second wave of signaling independent of G proteins. Thyroid-stimulating hormone receptor (TSHR) is one of the GPCR members. In our previous study, TSHR was identified in the liver; the major role of TSHR in cholesterol metabolism was illustrated, as TSH could regulate hepatic cholesterol metabolism via cAMP/PKA/CREB/HMGCR and SREBP2/HNF4α/CYP7A1 pathways. It has been reported that ARRB2 predominates over ARRB1 in TSHR internalization. However, the significance of ARRBs in TSH-initiated cholesterol metabolism has not been illustrated. In our study, the effects of ARRBs on TSH-regulated cholesterol metabolism are investigated. ARRB1/2 was genetically inactivated in C57BL/6 mice and HepG2 cell line, respectively. Cholesterol levels in arrestin-knockout mice and arrestin-knockdown cells were measured. Molecules participating in cholesterol metabolism were analyzed. It turned out that deficiencies in ARRB1 led to decreased cholesterol levels and decreased TSH-stimulated AKT phosphorylation. Subsequently, the inhibitory effect on CYP7A1 by SREBP2 was reduced due to lowered mature SREBP2 level. Other than the failures of TSH in ARRB-knockdown cells, the AKT activator SC79 could enhance AKT phosphorylation and mature SREBP2 level. Our results demonstrate that ARRBs, especially ARRB1, are involved in TSH-regulated cholesterol metabolism through the AKT pathway.

Transcriptome Analysis Reveals That G Protein-Coupled Receptors Are Potential Diagnostic Markers or Therapeutic Targets in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is associated with poor overall survival and the development of more effective therapies is urgently needed. G Protein-Coupled Receptors (GPCRs) represent the largest family of membrane receptors, with an estimated 800 members in humans, and are attractive therapeutic targets, accounting for approximately 30% of targets of marketed drugs. These receptors are key transducers that bind a vast diversity of ligands (e.g. glycoproteins, peptides, amino acids, nucleotides, nucleosides, ions) allowing the cells to adapt to their environment by regulating a wide variety of physiological processes including the control of blood pressure, heart rate, digestive processes, hormone secretion, cell growth and migration, as well as vision and olfaction. Binding to their ligands leads to conformational rearrangements promoting the engagement and modulation of many distinct downstream signaling effectors that are both G protein-dependent and independent.Several GPCRs are critical for cell proliferation and survival, and can be aberrantly expressed in cancer cells. For example, the chemokine receptor CXCR4 plays an important role in metastasis and angiogenesis in breast cancer and many other types of tumors. In AML, CXCR4 overexpression has been associated with poor outcome. Moreover, in vivo mouse studies have shown that the use of a small molecule antagonist of CXCR4 increases the mobilization of AML cells into the peripheral blood and improves the apoptotic effects of chemotherapy. This activity has been explored in a phase I/II clinical study showing that the addition of CXCR4 antagonists to chemotherapy in AML might improve the remission rate. The role of GPCRs in mouse leukemogenesis has also been suggested in a transcriptome analysis of two related leukemia clones that differ in their stem cell frequency. In this study, GPCRs were the most differentially expressed class of genes between the two clones.Currently, an extensive assessment of GPCR expression in human AML is lacking. To address this issue, we studied the expression of GPCRs in a large cohort of AML samples, as well as in normal blood cells, bone marrow cell populations, and cord blood-derived CD34+ cells as controls.The 772 GPCRs analyzed in this study consist of all the GPCR members included in the International Union of Basic and Clinical Pharmacology (IUPHAR) database, as well as 370 olfactory, 24 taste and 4 vomeronasal receptors. RNA sequencing data analysis was performed as previously described (Lavallée et al, Blood 2015 Jan 1;125(1):140-3) and revealed that 240 GPCRs are expressed in cells from this AML cohort. Among these receptors, 30 are upregulated and 19 are downregulated in AML samples compared to CD34+ normal cells. Upregulated GPCRs are enriched in chemokine (CCR1, CXCR4, CCR2, CX3CR1, CCR7, and CCRL2), adhesion (CD97, EMR1, EMR2, and GPR114) and purine (including P2RY2 and P2RY13) receptor sub-families. The downregulated members include adhesion GPCRs such as LPHN1, GPR125, GPR56, CELSR3, and GPR126, protease-activated receptors (F2R and F2RL1), and the Frizzled family receptors SMO and FZD6. Interestingly, specific deregulation was observed in genetically distinct subgroups of AML, a subset of GPCRs being differentially expressed in normal karyotype AML with NPM1 or FLT3 -ITD mutations, and in specimens with Core Binding Factor and MLL rearrangements, thereby representing promising therapeutic targets or diagnostic markers.In conclusion, our results demonstrate that several GPCR members are deregulated in AML with a clear enrichment in distinct classes, providing the rationale for functional assays using available agonists or antagonists to leukemia-enriched GPCRs. Since these receptors are the targets of several US Federal and Drug Administration approved drugs, our results pave the way to explore selected GPCRs as novel AML therapeutic targets. DisclosuresBouvier:American Society of Nephrology: Other: speaker; Domain Therapeutics: Other: Company SAB member, Research Funding; Vertex Pharmaceutical: Research Funding; Ontario Genomic Institute: Other: SAB meeting; BMS: Research Funding; DalCor Pharmaceutics: Other: Company SAB member; Pfizer: Other: Speaker, Research Funding; Novo-Nordisc: Research Funding.

High-level soluble expression of one model olfactory receptor (ODR-10) in Escherichia coli cell-free system

High-level production of G protein-coupled receptors (GPCRs) is usually difficult to achieve in heterologous cell systems. The inherent hydrophobicity of these receptors could cause aggregation and possible cytotoxicity. Cell-free (CF) expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. Here we reported the CF production of an olfactory receptor from Caenorhabditis elegans, odorant response abnormal protein 10 (ODR-10), a member of GPCRs, using the Escherichia coli extracts. Different expression vectors were investigated and 175μg/ml total ODR-10 was achieved with pIVEX2.4c. To obtain soluble ODR-10, different detergents and liposome with varied concentrations were respectively added into the CF system. High-level expression of soluble ODR-10 (150μg/ml) was attained with the addition of 1.5% polyoxyethylene-(20)-cetyl-ether (Brij58) into the CF system. Furthermore, the yield of total ODR-10 was improved to 350μg/ml by supplementing liposomes into the CF system, and the maximal concentration of the soluble receptor (102μg/ml) was achieved in this liposome-assisted CF system. Both strategies produced ODR-10 efficiently by using CF system, and the direct reconstitution of the in vitro expressed receptor into liposomes will be preferred for its potential applications in many areas.

Signaling property study of adhesion G-protein-coupled receptors

Adhesion G-protein-coupled receptors (GPCR) are special members of GPCRs with long N-termini containing multiple domains. We overexpressed our collection of receptors together with G-proteins in mammalian cell lines and measured the concentrations of intracellular signaling molecules, such as inositol phosphate and cAMP. Our results show that a subset of tested adhesion GPCRs has constitutive activities and is capable of coupling to a variety of G-proteins. In addition, we have identified a small molecule compound that specifically activates one of the subfamily members, GPR97, and the activation was confirmed by an independent GTPγS assay. These findings suggest classical GPCR screening assays could be applied to de-orphanize these receptors, and provide pharmacological tools to improve understanding of the physiological functions of these receptors.

Cross-talk and modulation of signaling between somatostatin and growth factor receptors

The process of homo- and/or heterodimerization of G-protein coupled receptors (GPCRs) and receptor tyrosine kinase (RTK) families are crucial for implicating the fundamental properties of receptor proteins including receptor expression, trafficking, and desensitization as well as signal transduction. The members of GPCR and RTK family constitute largest cell surface receptor proteins and regulate physiological functions of cells in response to external and internal stimuli. Notably, GPCRs and RTKs play major role in regulation of several key cellular functions which are associated with several pathological conditions including cancer biology, neurodegenerative and cardiovascular diseases. The focus of this review is to highlight the recent findings on the possible cross-talk between somatostatin receptors (members of GPCR family) and growth factor receptors like epidermal growth factor receptors (members of RTK family). Furthermore, functional consequences of such an interaction in modulation of signaling pathways linked to pathological conditions specifically in cancer are discussed.

G protein-coupled receptors: novel targets for drug discovery in cancer

G protein-coupled receptors (GPCRs) belong to a superfamily of cell surface signalling proteins that have a pivotal role in many physiological functions and in multiple diseases, including the development of cancer and cancer metastasis. Current drugs that target GPCRs - many of which have excellent therapeutic benefits - are directed towards only a few GPCR members. Therefore, huge efforts are currently underway to develop new GPCR-based drugs, particularly for cancer. We review recent findings that present unexpected opportunities to interfere with major tumorigenic signals by manipulating GPCR-mediated pathways. We also discuss current data regarding novel GPCR targets that may provide promising opportunities for drug discovery in cancer prevention and treatment.

Adenosine A2A Receptor Is Involved in Cell Surface Expression of A2B Receptor

The A2A and A2B adenosine receptors (A2AR and A2BR) are implicated in many physiological processes. However, the mechanisms of their intracellular maturation and trafficking are poorly understood. In comparative studies of A2AR versus A2BR expression in transfected cells, we noticed that the levels of cell surface expression of A2BR were significantly lower than those of A2AR. A large portion of the A2BR was degraded by the proteasome. Studies of cell surface expression of A2BR chimeric molecules in transfectants suggested that A2BR does not have the dominant forward transport signal for export from the endoplasmic reticulum to the cell surface. A2BR surface expression was increased in A2BR chimeras where the A2BR carboxyl terminus (CT) was replaced or fused with the A2AR CT. Co-transfection of A2AR with A2BR enhanced surface expression of A2BR though the F(X)(6)LL motif in the A2AR CT. The requirements of A2AR expression for better A2BR cell surface expression was not only established in transfectants but also confirmed by observations of much lower levels of A2BR-induced intracellular cAMP accumulation in response to A2BR-activating ligand in splenocytes from A2AR(-/-) mice than in wild type mice. The results of mechanistic studies suggested that poor A2BR expression at the cell surface might be accounted for mainly by the lack of a dominant forward transport signal from the endoplasmic reticulum to the plasma membrane; it is likely that A2BR forms a hetero-oligomer complex for better function.

GrlJ, a Dictyostelium GABAB-like receptor with roles in post-aggregation development

BackgroundThe G-protein-coupled receptor (GPCR) family represents the largest and most important group of targets for chemotherapeutics. They are extremely versatile receptors that transduce signals as diverse as biogenic amines, purins, odorants, ions and pheromones from the extracellular compartment to the interior via biochemical processes involving GTP-binding proteins. Until recently, the cyclic AMP receptors (cARs) were the only known G protein coupled receptors in Dictyostelium discoideum. The completed genome sequence revealed the presence of several families of GPCRs in Dictyostelium, among them members of the family 3 of GPCRs, the GABAB/glutamate like receptor family, which in higher eukaryotes is involved in neuronal signaling.ResultsD. discoideum has seventeen Family 3 members of GPCRs, denoted GrlA through GrlR. Their transcripts are detected throughout development with increased levels during early and late development. We have examined here GrlJ. GFP-tagged GrlJ localises to the plasmamembrane and to internal membranes. Inactivation of the grlJ gene leads to precocious development, and the mutant completes development ~6 hours earlier. Alterations were also noted at the slug stage and in spore formation. grlJ- slugs were longer and broke apart several times on their way to culmination forming smaller but proportionate fruiting bodies. Spores from grlJ- fruiting bodies were malformed and less viable, although the spore differentiation factors were synthesized and sensed normally. Expression of a GFP-tagged full length GrlJ rescued the phenotype.ConclusionOur data suggest that GrlJ acts at several stages of Dictyostelium development and that it is a negative regulator in Dictyostelium development.

Evolution of secretin family GPCR members in the metazoa

BackgroundComparative approaches using protostome and deuterostome data have greatly contributed to understanding gene function and organismal complexity. The family 2 G-protein coupled receptors (GPCRs) are one of the largest and best studied hormone and neuropeptide receptor families. They are suggested to have arisen from a single ancestral gene via duplication events. Despite the recent identification of receptor members in protostome and early deuterostome genomes, relatively little is known about their function or origin during metazoan divergence. In this study a comprehensive description of family 2 GPCR evolution is given based on in silico and expression analyses of the invertebrate receptor genes.ResultsFamily 2 GPCR members were identified in the invertebrate genomes of the nematodes C. elegans and C. briggsae, the arthropods D. melanogaster and A. gambiae (mosquito) and in the tunicate C. intestinalis. This suggests that they are of ancient origin and have evolved through gene/genome duplication events. Sequence comparisons and phylogenetic analyses have demonstrated that the immediate gene environment, with regard to gene content, is conserved between the protostome and deuterostome receptor genomic regions. Also that the protostome genes are more like the deuterostome Corticotrophin Releasing Factor (CRF) and Calcitonin/Calcitonin Gene-Related Peptide (CAL/CGRP) receptors members than the other family 2 GPCR members. The evolution of family 2 GPCRs in deuterostomes is characterised by acquisition of new family members, with SCT (Secretin) receptors only present in tetrapods. Gene structure is characterised by an increase in intron number with organismal complexity with the exception of the vertebrate CAL/CGRP receptors.ConclusionThe family 2 GPCR members provide a good example of gene duplication events occurring in tandem with increasing organismal complexity during metazoan evolution. The putative ancestral receptors are proposed to be more like the deuterostome CAL/CGRP and CRF receptors and this may be associated with their fundamental role in calcium regulation and the stress response, both of which are essential for survival.