Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The characteristics of inhibitory regulation of adenylyl cyclase V/VI by Ca2+ and G proteins were examined in dispersed gastric smooth muscle cells. The mechanisms were evoked separately, sequentially, or concurrently using ligand-gated and G protein-coupled receptor agonists and receptor-independent probes (e. g, thapsigargin). During the initial phase of agonist stimulation, alpha,beta-methylene-ATP, UTP, and ATP inhibited forskolin-stimulated cAMP formation in a concentration-dependent fashion. Inhibition by alpha,beta-methylene-ATP, which activates ligand-gated P2X receptors, was abolished by zero Ca2+, whereas inhibition by UTP, which activates P2Y2 receptors coupled to Gq/11 and Gi3, was not affected by zero Ca2+ but was abolished by pertussis toxin (PTX). Inhibition by ATP, which activates both P2X and P2Y2 receptors, was not affected by zero Ca2+ alone; but after inhibition mediated by Galphai3 was blocked with PTX, inhibition by Ca2+ influx was unmasked and was abolished by zero Ca2+. Inhibition by cholecystokinin-8 was observed only during the phase of capacitative Ca2+ influx and was blocked by zero Ca2+. Inhibition by UTP during this phase was not affected by zero Ca2+ alone; but after inhibition mediated by Galphai3 was blocked with PTX, inhibition by Ca2+ influx was unmasked and was abolished by zero Ca2+. Inhibition of adenylyl cyclase V/VI activity in smooth muscle can be mediated independently by inhibitory G proteins and Ca2+ influx but is exclusively mediated by inhibitory G proteins when both mechanisms are triggered.
Mol Pharmacol 1998 Jul
PMID:Regulation of adenylyl cyclase type V/VI in smooth muscle: interplay of inhibitory G protein and Ca2+ influx. 965 97

Modification of an S. cerevisiae G protein-coupled receptor with ubiquitin is required for its ligand-stimulated internalization. We now demonstrate that monoubiquitination on a single lysine residue is sufficient to signal receptor internalization, a modification distinct from that required for proteasome recognition. Formation of a polyubiquitin chain is not necessary, as demonstrated by the ability of mutant ubiquitins that lack lysine residues to serve as efficient internalization signals. Fusion of ubiquitin in-frame to a receptor that lacks cytoplasmic tail lysines also promotes rapid receptor internalization, indicating that ubiquitin itself and not a specific type of linkage to the receptor acts as an internalization signal. Thus, we have defined a cellular function for monoubiquitination in alpha-factor receptor endocytosis.
Mol Cell 1998 Jan
PMID:A function for monoubiquitination in the internalization of a G protein-coupled receptor. 965 16

Reorganization of the actin cytoskeleton is an early cellular response to a variety of extracellular signals. Dissection of pathways leading to actin rearrangement has focused largely on those initiated by growth factor receptors or integrins, although stimulation of G protein-coupled receptors also leads to cytoskeletal changes. In transfected Cos-7SH cells, activation of the chemoattractant formyl peptide receptor induces cortical actin polymerization and a decrease in the number of central actin bundles. In this report, we show that cytoskeletal reorganization can be transduced by G protein betagamma heterodimers (Gbetagamma), phosphoinositide 3-kinase gamma (PI3-Kgamma), a guanosine exchange factor (GEF) for Rac, and Rac. Expression of inactive variants of either PI3-Kgamma, the Rac GEF Vav, or Rac blocked the actin rearrangement. Neither wortmannin nor LY294002, pharmacologic inhibitors of PI3-K, could inhibit the actin rearrangement induced by a constitutively active Rac. The inhibition of cytoskeletal reorganization by the dominant negative Vav variants could be rescued by coexpression of a constitutively active form of Rac. In contrast, a Vav variant with its pleckstrin homology (PH) domain missing constitutively induced JNK activation and led to cytoskeletal reorganization, even without stimulation by PI3-Kgamma. This suggests that the PH domain of Vav controls the guanosine exchange activity of Vav, perhaps by a mechanism regulated by D3 phosphoinositides generated by PI3-K. Taken together, these findings delineate a pathway leading from activation of a G protein-coupled receptor to actin reorganization which sequentially involves Gbetagamma, PI3-Kgamma, a Rac GEF, and Rac.
Mol Cell Biol 1998 Aug
PMID:Cytoskeletal reorganization by G protein-coupled receptors is dependent on phosphoinositide 3-kinase gamma, a Rac guanosine exchange factor, and Rac. 967 84

The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G protein-coupled receptors. We have used homology genomic library screening and polymerase chain reaction (PCR) techniques to isolate both genomic and cDNA clones encoding the human homolog of the recently cloned rat GALR2 galanin receptor. By fluorescence in situ hybridization, the gene encoding human GALR2 (GALNR2) has been localized to chromosome 17q25.3. The two coding exons of the human GALNR2 gene, interrupted by an intron positioned at the end of transmembrane domain III, encode a 387 amino acid G protein-coupled receptor with 87% overall amino acid identity with rat GALR2. In HEK-293 cells stably expressing human GALR2, binding of [125I]porcine galanin is saturable and can be displaced by galanin, amino-terminal galanin fragments and chimeric galanin peptides but not by carboxy-terminal galanin fragments. In HEK-293 cells, human GALR2 couples both to Galphaq/11 to stimulate phospholipase C and increase intracellular calcium levels and to Galphai/o to inhibit forskolin-stimulated intracellular cAMP accumulation. A wide tissue distribution is observed by reverse transcriptase (RT)-PCR analysis, with human GALR2 mRNA being detected in many areas of the human central nervous system as well as in peripheral tissues.
Brain Res Mol Brain Res 1998 Jul 15
PMID:Molecular characterization, pharmacological properties and chromosomal localization of the human GALR2 galanin receptor. 968 25

Parathyroid hormone (PTH) and PTH-related protein interact with a G protein-coupled receptor linked to the activation of adenylyl cyclase and phospholipase C signaling pathways. Regulation by PTH of the expression of three distinct, stably transfected luciferase reporter genes responsive to cAMP (CRE-luc), serum (SRE-luc) and phorbol ester (TRE-luc) has been studied in rat osteoblast-like UMR-106 cells. Maximal 43-fold induction of CRE-luc expression occurred in response to 100 nM rat (r)PTH(1-34) (EC50=0.44 nM), but SRE-luc and TRE-luc remained unaffected. Maximal 2.8- and 3.4-fold inductions of SRE-luc by 10 ng/ml EGF and 100 nM phorbol ester (PMA) were suppressed with 100 nM rPTH(1-34) (IC50=0.04 and 0.15 nM, respectively). Similarly, 7.3-fold induction of TRE-luc by 100 nM PMA was inhibited to 50% with 100 nM rPTH(1-34) (IC50=0.5 nM). Activation of mitogen-activated protein kinase by EGF and PMA was also suppressed by rPTH(1-34). 1 mM 8-Br-cAMP and 0.1 mM forskolin mimicked all the effects of rPTH(1-34). In conclusion, the regulation of target genes by PTH in osteoblast-like UMR-106 cells is mediated by the activation of the cAMP/protein kinase A signaling pathway.
Mol Cell Endocrinol 1998 Apr 30
PMID:Parathyroid hormone responses of cyclic AMP-, serum- and phorbol ester-responsive reporter genes in osteoblast-like UMR-106 cells. 970 77

The study of G protein-coupled receptor signal transduction and behavior in living cells is technically difficult because of a lack of useful biological reagents. We show here that a fully functional alphalb-adrenoceptor tagged with the green fluorescent protein (alphalbAR/GFP) can be used to determine the molecular mechanism of intemalization of alphalbAR/ GFP in living cells. In mouse alphaT3 cells, alpha1bAR/GFP demonstrates strong, diffuse fluorescence along the plasma membrane when observed by confocal laser scanning microscope. The fluorescent receptor binds agonist and antagonist and stimulates phosphatidylinositol/Ca2+ signaling in a similar fashion to the wild receptor. In addition, alpha1bAR/ GFP can be internalized within minutes when exposed to agonist, and the subcellular redistribution of this receptor can be determined by measurement of endogenous fluorescence. The phospholipase C inhibitor U73,122, the protein kinase C activator PMA, and inhibitor staurosporine, and the Ca2+-ATPase inhibitor thapsigargin were used to examine the mechanism of agonist-promoted alphalbAR/GFP redistribution. Agonist-promoted internalization of alphalbAR/GFP was closely linked to phospholipase C activation and was dependent on protein kinase C activation, but was independent of the increase in intracellular free Ca2+ concentration. This study demonstrated that real-time optical monitoring of the subcellular localization of alphalbAR (as well as other G protein-coupled receptors) in living cells is feasible, and that this may provide a valuable system for further study of the biochemical mechanism(s) of agonist-induced receptor endocytosis.
Mol Endocrinol 1998 Aug
PMID:Real-time optical monitoring of ligand-mediated internalization of alpha1b-adrenoceptor with green fluorescent protein. 971 36

Little is known concerning the intracellular transport of the G protein-coupled receptors (GPCRs). Previous studies suggested a functional role for those residues immediately preceding the conserved palmitoylated cysteine residues in the intracellular carboxyl termini of some GPCRs in cell surface transport. For the human vasopressin V2 receptor, we assessed the significance of a dileucine sequence with an upstream glutamate residue (ELRSLLCC) in mediating cell surface delivery. A series of deletion and point mutants in this region were constructed, and the mutant receptors were expressed in transiently transfected COS.M6 cells. By using [3H]arginine vasopressin binding assays to intact cells and immunofluorescence studies with intact and permeabilized cells, we show that residues E335 (mutant E335Q) and L339 (mutant L339T) are obligatory for receptor transport to the plasma membrane. Residue L340 has a minor but significant influence. [3H]Arginine vasopressin binding experiments on membranes of lysed cells failed to detect any intracellular binding sites for the transport-deficient mutant receptors, suggesting that residues E335 and L339 participate in receptor folding. Studies with green fluorescent protein-tagged receptors demonstrate that the bulk of the mutant receptors E335Q and L339T are trapped in the endoplasmic reticulum. Complex glycosylation was absent in these mutant receptors, supporting this conclusion. These data demonstrate that the glutamate/dileucine motif of the vasopressin V2 receptor is critical for the escape of the receptor from the endoplasmic reticulum, most presumably by establishing a functional and transport-competent folding state. A databank analysis revealed that these residues are part of a conserved region in the GPCR family.
Mol Pharmacol 1998 Sep
PMID:A dileucine sequence and an upstream glutamate residue in the intracellular carboxyl terminus of the vasopressin V2 receptor are essential for cell surface transport in COS.M6 cells. 973 Sep 11

The V2 vasopressin renal receptor (V2R), which controls antidiuresis in mammals, is a member of the large family of heptahelical transmembrane (7TM) G protein-coupled receptors (GPCRs). Using the automated GPCR modeling facility available via Internet (http:/(/)expasy.hcuge.ch/swissmod/SWISS-MODEL.+ ++html) for construction of the 7TM domain in accord with the bovine rhodopsin (RD) footprint, and the SYBYL software for addition of the intra- and extracellular domains, the human V2R was modeled. The structure was further refined and its conformational variability tested by the use of a version of the Constrained Simulated Annealing (CSA) protocol developed in this laboratory. An inspection of the resulting structure reveals that the V2R (likewise any GPCR modeled this way) is much thicker and accordingly forms a more spacious TM cavity than most of the hitherto modeled GPCR constructs do, typically based on the structure of bacteriorhodopsin (BRD). Moreover, in this model the 7TM helices are arranged differently than they are in any BRD-based model. Thus, the topology and geometry of the TM cavity, potentially capable of receiving ligands, is in this model quite different than it is in the earlier models. In the subsequent step, two ligands, the native [arginine8]vasopressin (AVP) and the selective agonist [D-arginine8]vasopressin (DAVP) were inserted, each in two topologically non-equivalent ways, into the TM cavity and the resulting structures were equilibrated and their conformational variabilities tested using CSA as above. The best docking was selected and justified upon consideration of ligand-receptor interactions and structure-activity data. Finally, the amino acid residues were indicated, mainly in TM helices 3-7, as potentially important in both AVP and DAVP docking. Among those Cys112, Val115-Lys116, Gln119, Met123 in helix 3; Glu174 in helix 4; Val206, Ala210, Val213-Phe214 in helix 5; Trp284, Phe287-Phe288, Gln291 in helix 6; and Phe307, Leu310, Ala314 and Asn317 in helix 7 appeared to be the most important ones. Many of these residues are invariant for either the GPCR superfamily or the neurophyseal (vasopressin V2R, V1aR and V1bR and oxytocin OR) subfamily of receptors. Moreover, some of the equivalent residues in V1aR have already been found critical for the ligand affinity.
J Comput Aided Mol Des 1998 May
PMID:Molecular modeling of the human vasopressin V2 receptor/agonist complex. 974 70

In Dictyostelium discoideum, a unique Gbeta subunit is required for a G protein-coupled receptor system that mediates a variety of cellular responses. Binding of cAMP to cAR1, the receptor linked to the G protein G2, triggers a cascade of responses, including activation of adenylyl cyclase, gene induction, actin polymerization, and chemotaxis. Null mutations of the cAR1, Galpha2, and Gbeta genes completely impair all these responses. To dissect specificity in Gbetagamma signaling to downstream effectors in living cells, we screened a randomly mutagenized library of Gbeta genes and isolated Gbeta alleles that lacked the capacity to activate some effectors but retained the ability to regulate others. These mutant Gbeta subunits were able to link cAR1 to G2, to support gene expression, and to mediate cAMP-induced actin polymerization, and some were able to mediate to chemotaxis toward cAMP. None was able to activate adenylyl cyclase, and some did not support chemotaxis. Thus, we separated in vivo functions of Gbetagamma by making point mutations on Gbeta. Using the structure of the heterotrimeric G protein displayed in the computer program CHAIN, we examined the positions and the molecular interactions of the amino acids substituted in each of the mutant Gbetas and analyzed the possible effects of each replacement. We identified several residues that are crucial for activation of the adenylyl cyclase. These residues formed an area that overlaps but is not identical to regions where bovine Gtbetagamma interacts with its regulators, Galpha and phosducin.
Mol Biol Cell 1998 Oct
PMID:Selection of gbeta subunits with point mutations that fail to activate specific signaling pathways in vivo: dissecting cellular responses mediated by a heterotrimeric G protein in Dictyostelium discoideum. 976 54

We have previously shown that phosphatidylinositol 3-kinase alpha (PI 3-Kalpha) (p85alpha-p110alpha) is required for DNA synthesis induced by various growth factors (S. Roche, M. Koegl, and S. A. Courtneidge, Proc. Natl. Acad. Sci. USA 91:9185-9189, 1994) in fibroblasts. In the present study, we have investigated the function of PI 3-Kbeta (p85alpha-p110beta) during mitogenesis. By using antibodies specific to p110beta we showed that PI 3-Kbeta is expressed in NIH 3T3 cells. PI 3-Kbeta and PI 3-Kalpha have common features: PI 3-Kbeta is tightly associated with a protein serine kinase that phosphorylates p85alpha, it interacts with the Src-middle T antigen complex and the activated platelet-derived growth factor (PDGF) receptor in fibroblasts in vivo, and it becomes tyrosine phosphorylated after PDGF stimulation. PI 3-Kbeta was also activated in Swiss 3T3 and Cos7 cells stimulated with lysophosphatidic acid (LPA), a mitogen that interacts with a heterotrimeric G protein-coupled receptor. In contrast PI 3-Kalpha was activated to a lesser extent in these cells. Microinjection of neutralizing antibodies specific for p110beta into quiescent fibroblasts inhibited DNA synthesis induced by both insulin and LPA but poorly affected PDGF receptor signaling. Therefore, PI 3-Kbeta plays an important role in transmitting the mitogenic response induced by some, but not all, growth factors. Finally, we show that while oncogenic V12Ras interacts with type I PI 3-Ks, it could induce DNA synthesis in the absence of active PI 3-Kalpha and PI 3-Kbeta, suggesting that Ras uses other effectors for DNA synthesis.
Mol Cell Biol 1998 Dec
PMID:A function for phosphatidylinositol 3-kinase beta (p85alpha-p110beta) in fibroblasts during mitogenesis: requirement for insulin- and lysophosphatidic acid-mediated signal transduction. 981 98


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