Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “GPCR”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 955 records · Page 53Linked to original sources
PubMed2006

Adenylyl cyclase 6 overexpression decreases the permeability of endothelial monolayers via preferential enhancement of prostacyclin receptor function.

Overexpression of adenylyl cyclase (AC) has been proposed as a potential gene therapy strategy to increase cAMP formation in cardiomyocytes and cardiac function in vivo. The impact of AC overexpression on endothelial cells, which will be traversed by genes delivered in vivo, has not been examined. Hence, the goal of the current study was to determine the consequence of AC overexpression on vascular endothelial cells in terms of G-protein-coupled receptor (GPCR) signaling and endothelial barrier function. We demonstrate that adenoviral-mediated gene transfer of AC6 in human umbilical vein endothelial cells preferentially enhances prostacyclin receptor (versus other GPCR)-stimulated cAMP synthesis and, in parallel, inhibits thrombin-stimulated increases in endothelial cell barrier function. Using multiple strategies, including prostacyclin receptor-targeted small interfering RNA, we identify that the enhancement of endothelial barrier function by AC6 overexpression is dependent on an autocrine/paracrine feedback pathway involving the release of prostacyclin and activation of prostacyclin receptors. AC6 overexpression in endothelial cells may have use as a means to enhance prostacyclin function and reduce endothelial barrier permeability.

Adenoviridae↗
PubMed2006

Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay.

Regulators of G-protein signaling (RGS) proteins are important components of signal transduction pathways initiated through G-protein-coupled receptors (GPCRs). RGS proteins accelerate the intrinsic GTPase activity of G-protein alpha-subunits (Galpha) and thus shorten the time course and reduce the magnitude of G-protein alpha- and betagamma-subunit signaling. Inhibiting RGS action has been proposed as a means to enhance the activity and specificity of GPCR agonist drugs, but pharmacological targeting of protein-protein interactions has typically been difficult. The aim of this project was to identify inhibitors of RGS4. Using a Luminex 96-well plate bead analyzer and a novel flow-cytometric protein interaction assay to assess Galpha-RGS interactions in a high-throughput screen, we identified the first small-molecule inhibitor of an RGS protein. Of 3028 compounds screened, 1, methyl N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986), inhibited RGS4/Galpha(o) binding with 3 to 5 muM potency. It binds to RGS4, inhibits RGS4 stimulation of Galpha(o) GTPase activity in vitro, and prevents RGS4 regulation of mu-opioid-inhibited adenylyl cyclase activity in permeabilized cells. Furthermore, CCG-4986 is selective for RGS4 and does not inhibit RGS8. Thus, we demonstrate the feasibility of targeting RGS/Galpha protein-protein interactions with small molecules as a novel means to modulate GPCR-mediated signaling processes.

Biotinylation↗
PubMed2006

The highly conserved DRY motif of class A G protein-coupled receptors: beyond the ground state.

Despite extensive study of heptahelical G protein-coupled receptors (GPCRs), the precise mechanism of G protein activation is unknown. The role of one highly conserved stretch of residues, the amino acids glutamic acid/aspartic acid-arginine-tyrosine (i.e., the E/DRY motif), has received considerable attention with respect to regulating GPCR conformational states. In the consensus view, glutamic acid/aspartic acid maintains the receptor in its ground state, because mutations frequently induce constitutive activity (CA). This hypothesis has been confirmed by the rhodopsin ground-state crystal structure and by computational modeling approaches. However, some class A GPCRs are resistant to CA, suggesting alternative roles for the glutamic acid/aspartic acid residue and the E/DRY motif. Here, we propose two different subgroups of receptors within class A GPCRs that make different use of the E/DRY motif, independent of the G protein type (G(s), G(i), or G(q)) to which the receptor couples. In phenotype 1 receptors, nonconservative mutations of the glutamic acid/aspartic acid-arginine residues, besides inducing CA, increase affinity for agonist binding, retain G protein coupling, and retain an agonist-induced response. In contrast, in second phenotype receptors, the E/DRY motif is more directly involved in governing receptor conformation and G protein coupling/recognition. Hence, mutations of the glutamic acid/aspartic acid residues do not induce CA. Conversely, nonconservative mutations of the arginine of the E/DRY motif always impair agonist-induced receptor responses and, generally, reduce agonist binding affinity. Thus, it is essential to look beyond the rhodopsin ground-state model of conformational activation to clarify the role of this highly conserved triplet in GPCR activation and function.

Amino Acid Motifs↗
PubMed1997

Naloxone activation of mu-opioid receptors mutated at a histidine residue lining the opioid binding cavity.

The mu-opioid receptor is the principal site of action in the brain by which morphine, other opiate drugs of abuse, and endogenous opioid peptides effect analgesia and alter mood. A member of the seven-transmembrane domain (TM) G protein-coupled receptor (GPCR) superfamily, the mu-opioid receptor modulates ion channels and second messenger effectors in an opioid agonist-dependent fashion that is reversible by the classic opiate antagonist naloxone. Mutation of a histidine residue (His297) in TM 6 afforded agonist-like G protein-coupled signal transduction mediated by naloxone and other alkaloid antagonists and enhanced the intrinsic activity of documented alkaloid partial agonists, including buprenorphine. The intrinsic activities of all opioid peptide agonists and antagonists tested were not altered at the His297 mutant receptors. Consistent with a role for the TM 6 histidine in maintaining high affinity binding sites for opioid agonists and antagonists, opioid ligand-dependent protection of this residue from a histidine-specific alkylating agent indicated that the His297 side chain is positioned in or very near the binding cavity. The TM 6 His297 mutants identify a discrete region of the receptor critical for determining whether a specific drug pharmacophore triggers receptor activation. Because many GPCRs possess a similarly positioned TM histidine residue, our findings with the mu-opioid receptor may extend to these receptors and potentially serve as a model for rational design of therapeutic GPCR partial agonists and antagonists.

Alkaloids↗
PubMed2001

The role of a conserved inter-transmembrane domain interface in regulating alpha(2a)-adrenergic receptor conformational stability and cell-surface turnover.

Functional and structural data from G protein-coupled receptors (GPCR) predict that transmembrane-domain (TM)2 is adjacent to TM7 within the GPCR structure, and that within this interface a conserved aspartate in TM2 and a conserved asparagine in TM7 exist in close proximity. Mutation at this D79(TM2)-N422(TM7) interface in the alpha(2A)-adrenergic receptor (alpha(2A)AR) affects not only receptor activation but also cell-surface residence time and conformational stability. Mutation at TM2(D79N) reduces allosteric modulation by Na(+) and receptor activation more dramatically than affecting cell-surface receptor turnover and conformational stability, whereas mutation at TM7(N422D) creates profound conformational instability and more rapid degradation of receptor from the surface of cells despite receptor activation and allosteric modulation properties that mirror a wild-type receptor. Double mutation of TM2 and 7(D79N/N422D) reveals phenotypes for receptor activation and conformational stability intermediate between the wild-type and singly mutated alpha(2A)AR. Additionally, the structural placement of a negative charge at this TM2/TM7 interface is necessary but not sufficient for receptor structural stability, because mislocalization of the negative charge in either the D79E alpha(2A)AR (which extends the charge out one methylene group) or the D79N/N422D alpha(2A)AR (placing the charge in TM7 instead of TM2) results in conformational lability in detergent solution and more rapid cell-surface receptor clearance. These studies suggest that this interface is important in regulating receptor cell-surface residence time and conformational stability in addition to its previously recognized role in receptor activation.

Allosteric Regulation↗
PubMed2001

Agonist-stimulated and tonic internalization of metabotropic glutamate receptor 1a in human embryonic kidney 293 cells: agonist-stimulated endocytosis is beta-arrestin1 isoform-specific.

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that contribute to the regulation of integrative brain functions such as cognition, motor control, and neural development. Metabotropic glutamate receptors are members of a unique class of GPCRs (class III) that include the calcium sensing and gamma-aminobutyric acid type B receptors. Although mGluRs bear little sequence homology to well-characterized members of the GPCR superfamily, both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs) contribute to mGluR desensitization. Therefore, in the present study, we examined whether beta-arrestins, regulators of GPCR desensitization and endocytosis, are required for mGluR1a desensitization and internalization in human embryonic kidney (HEK) 293 cells. Unlike what has been reported for other GPCRs, we find that in response to agonist stimulation, mGluR1a internalization is selectively mediated by beta-arrestin1 in HEK 293 cells. However, even though beta-arrestin1 binds directly to the carboxyl-terminal tail of mGluR1a and redistributes with mGluR1a to endosomes, neither beta-arrestin1 nor beta-arrestin2 seems to contribute to mGluR1a desensitization in HEK 293 cells. We also observed extensive tonic mGluR1a internalization via clathrin-coated vesicles in the absence of agonist. The tonic internalization of mGluR1a is insensitive to antagonist treatment, dominant-negative mutants of GRK2, beta-arrestin1, and dynamin as well as treatments that disrupt caveolae, but is blocked by hypertonic sucrose and concanavalin A treatment. Internalized mGluR1a is colocalized with clathrin, transferrin receptor, beta2-adrenergic receptor, and Rab5 GTPase in endocytic vesicles. Therefore, although mGluR1a internalizes with beta-arrestin in response to agonist, the agonist-independent internalization of mGluR1a involves the beta-arrestin-independent targeting of mGluR1a to clathrin-coated vesicles.

Arrestins↗
PubMed2003

The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints.

The superfamily of G-protein-coupled receptors (GPCRs) is very diverse in structure and function and its members are among the most pursued targets for drug development. We identified more than 800 human GPCR sequences and simultaneously analyzed 342 unique functional nonolfactory human GPCR sequences with phylogenetic analyses. Our results show, with high bootstrap support, five main families, named glutamate, rhodopsin, adhesion, frizzled/taste2, and secretin, forming the GRAFS classification system. The rhodopsin family is the largest and forms four main groups with 13 sub-branches. Positions of the GPCRs in chromosomal paralogons regions indicate the importance of tetraploidizations or local gene duplication events for their creation. We also searched for "fingerprint" motifs using Hidden Markov Models delineating the putative inter-relationship of the GRAFS families. We show several common structural features indicating that the human GPCRs in the GRAFS families share a common ancestor. This study represents the first overall map of the GPCRs in a single mammalian genome. Our novel approach of analyzing such large and diverse sequence sets may be useful for studies on GPCRs in other genomes and divergent protein families.

Chromosome Mapping↗
PubMed2003

Membrane assembly of the cannabinoid receptor 1: impact of a long N-terminal tail.

The human cannabinoid receptor 1 (CB1) belongs to the G protein-coupled receptor (GPCR) family. Among the members of GPCR family, it has an exceptionally long extracellular N-terminal domain (N-tail) of 116 amino acids but has no typical signal sequence. This poses questions of how the long N-tail affects the biosynthesis of the receptor and of how it is inserted into the endoplasmic reticulum (ER) membrane. Here we have examined the process of membrane assembly of CB1 in the ER membrane and the maturation of the receptor from the ER to the plasma membrane. We find that the long N-tail cannot be efficiently translocated across the ER membrane, causing the rapid degradation of CB1 by proteasomes; this leads to a low level of expression of the receptor at the plasma membrane. The addition of a signal peptide at the N terminus of CB1 or shortening of the long N-tail greatly enhances the stability and cell surface expression of the receptor without affecting receptor binding to a cannabinoid ligand, CP-55,940. We propose that the N-tail translocation is a crucial early step in biosynthesis of the receptor and may play a role in regulating the stability and surface expression of CB1.

Animals↗
PubMed2003

Involvement of intramolecular interactions in the regulation of G protein-coupled receptor kinase 2.

The G protein-coupled receptor (GPCR) kinase GRK2 phosphorylates G protein-coupled receptors in an agonist-dependent manner. GRK2 activity is modulated through interactions of diverse domains of the kinase with G protein betagamma subunits, several lipids, anchoring proteins, and activated receptors. We report that kinase activity toward either GPCR (rhodopsin) or a synthetic peptide substrate is enhanced in the presence of GST-GRK2 fusion proteins or peptides corresponding to either N- or C-terminal sequences of GRK2. This direct stimulatory action of intrinsic domains on GRK2 activity does not add to the effect of other regulators, such as Gbetagamma subunits, and strongly suggests the existence of some mode of autoregulation. The existence of regulatory intramolecular interactions in GRK2 is supported by the facts that a C-terminal peptide protects the N-terminal region from proteolytic cleavage and that two domains of GRK2 independently coexpressed in cells associate as assessed by immunoprecipitation. Molecular modeling suggests that intramolecular interactions among the N-terminal, C-terminal and kinase domains would keep GRK2 in a constrained conformation characteristic of an inactive, basal state. Our model proposes that disruption of such intramolecular contacts by intermolecular interactions with regulatory proteins (mimicked by exogenously added kinase fragments in vitro) would promote the conformational changes required to bring about GRK2 translocation and activation.

Animals↗
PubMed2004

Temporal characteristics of cAMP response element-mediated gene transcription: requirement for sustained cAMP production.

Many clinically used drugs are G-protein-coupled receptor (GPCR) antagonists and are given long-term to prevent receptor activation by endogenous agonists. Most GPCR antagonists are considered to have little agonist efficacy of their own. However, many beta antagonists do stimulate very small beta(2) adrenoceptor-mediated cAMP responses, but these responses become substantial at the level of cAMP response element (CRE)-gene transcription. Here, we compared the temporal characteristics of these beta(2) adrenoceptor-mediated cAMP and CRE-gene transcription responses with ligands of differing agonist efficacy. Within a minute, full agonists (e.g., isoprenaline) stimulated large increases in intracellular and exported cAMP. Very weak partial agonists (e.g., alprenolol) did not increase intracellular cAMP (only stimulating a small export). However, all agonists (regardless of efficacy) stimulated an increase in CRE-gene transcription after a 2-h incubation. An initial 30-min continual stimulation was required to initiate the process of CRE-gene transcription for all ligands. Longer agonist incubations resulted in larger gene transcription responses in a proportional manner for both weak and full agonists alike, and this was despite the lack of intracellular cAMP detection for the weaker ligands. Thus, the major initiator for CRE-gene transcription was not cAMP concentration or total quantity generated but a sustained turnover of intracellular cAMP and hence sustained stimulation of CREB phosphorylation. Thus, long-acting agonists and long-term treatments with very weak partial agonists (including many drugs classified previously as antagonists based on traditional second-messenger assays, e.g., several clinically used "beta-blockers") may cause more substantial gene transcription than previously believed.

Animals↗
PubMed2004

Agonist-induced internalization of leukotriene B(4) receptor 1 requires G-protein-coupled receptor kinase 2 but not arrestins.

The leukotriene B(4) (LTB(4)) receptor (BLT1) becomes desensitized upon repeated agonist stimulation. Little is known, however, about BLT1 internalization, which follows desensitization in most G-protein-coupled receptors (GPCR). In the current study, transiently expressed BLT1 readily internalized, after LTB(4) stimulation, in RBL-2H3 cells that express high levels of endogenous GPCR kinase 2 (GRK2) but did not in COS-7 or human embryonic kidney (HEK) 293 cells, which do not overexpress GRK. The internalization of BLT1 could be blocked in RBL-2H3 cells by coexpressing dominant-negative (DN) GRK2 K220R and could be promoted in HEK293 cells by coexpressing wild-type (WT) GRK2. Coexpression of WT or DN nonvisual arrestins had no effect on BLT1 internalization. Moreover, upon stimulation with LTB(4), BLT1 did not induce arrestin-green fluorescence protein redistribution in either cell type, even in the presence of overexpressed GRK2. Coimmunoprecipitation experiments confirmed that BLT1 could associate with GRK2 but not with arrestins. A C-tail-truncated mutant of BLT1 lost the capacity to internalize and associate with GRK2 upon exposure to LTB(4), suggesting that the C-tail was required for receptor internalization and association with GRK2. Taken together, our results indicate that the C terminus of BLT1 plays a pivotal role in receptor internalization and GRK2 association. Moreover, ligand-induced BLT1 internalization is dependent on GRK2 but independent of arrestins. This may allow differential, cell-type-specific signaling in response to LTB(4), depending on GRK expression levels.

Animals↗
PubMed2005

Heterodimerization of g protein-coupled receptors: specificity and functional significance.

G protein-coupled receptors (GPCRs) are cell surface receptors that mediate physiological responses to a diverse array of stimuli. GPCRs have traditionally been thought to act as monomers, but recent evidence suggests that GPCRs may form dimers (or higher-order oligomers) as part of their normal trafficking and function. In fact, certain GPCRs seem to have a strict requirement for heterodimerization to attain proper surface expression and functional activity. Even those GPCRs that do not absolutely require heterodimerization may still specifically associate with other GPCR subtypes, sometimes resulting in dramatic effects on receptor pharmacology, signaling, and/or internalization. Understanding the specificity and functional significance of GPCR heterodimerization is of tremendous clinical importance since GPCRs are the molecular targets for numerous therapeutic drugs.

Animals↗
PubMed2001

Role of the sphingosine-1-phosphate receptor EDG-1 in PDGF-induced cell motility.

EDG-1 is a heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) for sphingosine-1-phosphate (SPP). Cell migration toward platelet-derived growth factor (PDGF), which stimulates sphingosine kinase and increases intracellular SPP, was dependent on expression of EDG-1. Deletion of edg-1 or inhibition of sphingosine kinase suppressed chemotaxis toward PDGF and also activation of the small guanosine triphosphatase Rac, which is essential for protrusion of lamellipodia and forward movement. Moreover, PDGF activated EDG-1, as measured by translocation of beta-arrestin and phosphorylation of EDG-1. Our results reveal a role for receptor cross-communication in which activation of a GPCR by a receptor tyrosine kinase is critical for cell motility.

Animals↗
PubMed2001

Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin.

Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) function. Agonist stimulation of endogenous or transfected beta2-adrenergic receptors (beta2ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, beta-arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the beta2AR. Receptor ubiquitination required beta-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of beta-arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalization with marginal effects on receptor degradation. However, a beta2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of beta-arrestin in mediating the ubiquitination of the beta2AR and indicate that ubiquitination of the receptor and of beta-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.

Animals↗
PubMed2003

A seven-transmembrane RGS protein that modulates plant cell proliferation.

G protein-coupled receptors (GPCRs) at the cell surface activate heterotrimeric G proteins by inducing the G protein alpha (Galpha) subunit to exchange guanosine diphosphate for guanosine triphosphate. Regulators of G protein signaling (RGS) proteins accelerate the deactivation of Galpha subunits to reduce GPCR signaling. Here we identified an RGS protein (AtRGS1) in Arabidopsis that has a predicted structure similar to a GPCR as well as an RGS box with GTPase accelerating activity. Expression of AtRGS1 complemented the pheromone supersensitivity phenotype of a yeast RGS mutant, sst2Delta. Loss of AtRGS1 increased the activity of the Arabidopsis Galpha subunit, resulting in increased cell elongation in hypocotyls in darkness and increased cell production in roots grown in light. These findings suggest that AtRGS1 is a critical modulator of plant cell proliferation.

Alleles↗
PubMed2004

G protein-coupled receptor-dependent development of human frontal cortex.

The mammalian cerebral cortex is characterized by complex patterns of anatomical and functional areas that differ markedly between species, but the molecular basis for this functional subdivision is largely unknown. Here, we show that mutations in GPR56, which encodes an orphan G protein-coupled receptor (GPCR) with a large extracellular domain, cause a human brain cortical malformation called bilateral frontoparietal polymicrogyria (BFPP). BFPP is characterized by disorganized cortical lamination that is most severe in frontal cortex. Our data suggest that GPCR signaling plays an essential role in regional development of human cerebral cortex.

Amino Acid Substitution↗
PubMed2000

Function of PI3Kgamma in thymocyte development, T cell activation, and neutrophil migration.

Phosphoinositide 3-kinases (PI3Ks) regulate fundamental cellular responses such as proliferation, apoptosis, cell motility, and adhesion. Viable gene-targeted mice lacking the p110 catalytic subunit of PI3Kgamma were generated. We show that PI3Kgamma controls thymocyte survival and activation of mature T cells but has no role in the development or function of B cells. PI3Kgamma-deficient neutrophils exhibited severe defects in migration and respiratory burst in response to heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPCR) agonists and chemotactic agents. PI3Kgamma links GPCR stimulation to the formation of phosphatidylinositol 3,4,5-triphosphate and the activation of protein kinase B, ribosomal protein S6 kinase, and extracellular signal-regulated kinases 1 and 2. Thus, PI3Kgamma regulates thymocyte development, T cell activation, neutrophil migration, and the oxidative burst.

Animals↗
PubMed2000

Beta-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3.

beta-Arrestins, originally discovered in the context of heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) desensitization, also function in internalization and signaling of these receptors. We identified c-Jun amino-terminal kinase 3 (JNK3) as a binding partner of beta-arrestin 2 using a yeast two-hybrid screen and by coimmunoprecipitation from mouse brain extracts or cotransfected COS-7 cells. The upstream JNK activators apoptosis signal-regulating kinase 1 (ASK1) and mitogen-activated protein kinase (MAPK) kinase 4 were also found in complex with beta-arrestin 2. Cellular transfection of beta-arrestin 2 caused cytosolic retention of JNK3 and enhanced JNK3 phosphorylation stimulated by ASK1. Moreover, stimulation of the angiotensin II type 1A receptor activated JNK3 and triggered the colocalization of beta-arrestin 2 and active JNK3 to intracellular vesicles. Thus, beta-arrestin 2 acts as a scaffold protein, which brings the spatial distribution and activity of this MAPK module under the control of a GPCR.

Angiotensin II↗