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)

We have identified a putative opioid receptor from mouse brain (KOR-3), belonging to the G protein-coupled receptor family, that is distinct from the previously cloned mu, delta, and kappa 1 receptors. Assignment of the clone to the opioid receptor family derives from both structural and functional studies. Its predicted amino acid sequence is highly homologous to that of the other opioid receptors, particularly in many of the transmembrane regions, where long stretches are identical to mu, delta, and kappa 1 receptors. Both cyclazocine and nalorphine inhibit cAMP accumulation in COS-7 cells stably expressing the clone. Northern analysis shows that the mRNA is present in brain but not in a number of other organs. Southern analysis suggests a single gene encoding the receptor. A highly selective monoclonal antibody directed against the native kappa 3 receptor recognizes, in Western analysis, the clone expressed in COS-7 cells. The in vitro translation product is also labeled by the antibody. Additional clones reveal the presence of several introns, including one in the second extracellular loop and another in the first transmembrane region. Antisense studies with an oligodeoxynucleotide directed against a region of the second extracellular loop reveal a selective blockade of kappa 3 analgesia in vivo that is not observed with a mismatch oligodeoxynucleotide based upon the antisense sequence. The mu, delta, and kappa 1 analgesia is unaffected by this antisense treatment. Antisense mapping of the clone downstream from the splice site in the first transmembrane region reveals that six different antisense oligodeoxynucleotides all block kappa 3 analgesia. In contrast, only one of an additional six different antisense oligodeoxynucleotides directed at regions upstream from this splice site is effective. This strong demarcation between the two regions raises the possibility of splice variants of the receptor. An additional clone reveals an insert in the 3' untranslated region. In conclusion, the antibody and antisense studies strongly associate KOR-3 with the kappa 3-opioid receptor, although it is not clear whether it is the kappa 3 receptor itself or a splice variant.
Mol Pharmacol 1995 Jun
PMID:Cloning and functional characterization through antisense mapping of a kappa 3-related opioid receptor. 760 58

1. The cloning of the mammalian gonadotropin-releasing hormone receptor sets the stage for rapid progress in understanding the structure of the receptor, its interaction with ligand, and its mechanisms of activation. 2. The receptor is a 327 to 328-amino acid seven-transmembrane domain G protein-coupled receptor. 3. Recent site-direct mutagenesis studies have provided considerable insight into glycosylation of the receptor, the arrangement of the helices, and the ligand binding domains.
Cell Mol Neurobiol 1995 Feb
PMID:Functional domains of the gonadotropin-releasing hormone receptor. 764 8

With chronic opiate use, opioid receptor desensitization may be one of the important mechanisms underlying the development of opiate tolerance and addiction. Opioid receptors belong to the G protein-coupled receptor superfamily. In this study, the mouse delta-opioid receptor (delta OR) was used in a model system to investigate the role of opioid receptor phosphorylation in receptor desensitization. When expressed in 293 cells and exposed to agonist, the delta OR underwent receptor-specific desensitization within 10 min. This agonist-induced desensitization corresponded temporally to a 3-fold increase in receptor phosphorylation. Phorbol ester, but not forskolin, also stimulated phosphorylation of the delta OR in 293 cells. Although down-regulation of protein kinase C failed to affect agonist-induced receptor phosphorylation, it abolished phorbol ester-induced receptor phosphorylation. Agonist-induced delta OR phosphorylation must therefore involve kinases other than protein kinase C. Whereas overexpression of a dominant negative mutant (K220R) of beta-adrenergic receptor kinase-1 (beta ARK1) in 293 cells significantly reduced agonist-dependent phosphorylation of the delta OR, overexpression of beta ARK1 or G protein-coupled receptor kinase-5 significantly enhanced this phosphorylation. Concordantly, beta ARK1-K220R overexpression reduced agonist-dependent delta OR desensitization, whereas beta ARK1 overexpression enhanced this densensitization. We conclude that short term desensitization of the delta OR involves phosphorylation of the receptor by one or more G protein-coupled receptor kinases.
Mol Pharmacol 1995 Aug
PMID:Agonist-dependent phosphorylation of the mouse delta-opioid receptor: involvement of G protein-coupled receptor kinases but not protein kinase C. 765 49

The 5-hydroxytryptamine (5-HT)2C receptor is a G protein-coupled receptor that exhibits constitutive receptor activation, defined as agonist-independent receptor activation of the signal transduction pathway. The present studies were performed to determine whether NIH/3T3 fibroblasts expressing the 5-HT2C receptor exhibited desensitization of agonist-mediated phosphoinositide hydrolysis. Furthermore, 5-HT2C receptor-specific antibodies were used to determine whether the 5-HT2C receptor was phosphorylated in the absence of agonist and whether treatment with an agonist or an inverse agonist altered receptor phosphorylation. Time course studies of basal and serotonin-stimulated phosphoinositide hydrolysis demonstrated that basal values increased in a linear manner, whereas the response to serotonin plateaued within 60 min. In addition, pretreatment with serotonin resulted in a rightward shift of the subsequently determined serotonin dose-response curve. To determine the phosphorylation state of the 5-HT2C receptor, specific antibodies were used to immunoprecipitate the receptor from lysates prepared from 32P-labeled fibroblasts. Phosphorylation of the 5-HT2C receptor was evident under basal conditions, and serotonin treatment increased receptor phosphorylation. The inverse agonist mianserin had no detectable effect on 5-HT2C receptor phosphorylation when added alone but blocked the serotonin-stimulated increase in 5-HT2C receptor phosphorylation. The present study is the first to demonstrate that the 5-HT2C receptor is phosphorylated under basal conditions and phosphorylation is increased by agonist treatment conditions that result in desensitization of receptor signaling. Thus, these studies demonstrate that a cell line exhibiting a high level of constitutive 5-HT2C receptor activity has the ability to undergo agonist-mediated desensitization, consistent with current models of G protein-coupled receptor regulation.
Mol Pharmacol 1995 Aug
PMID:Increased basal phosphorylation of the constitutively active serotonin 2C receptor accompanies agonist-mediated desensitization. 765 52

The thyrotropin-releasing hormone (TRH) receptor (TRH-R) is a member of the seven-transmembrane region, G protein-coupled receptor family. Arg-283 and Arg-306, in transmembrane helices 6 and 7, respectively, are putatively in positions homologous to those of residues that are important for agonist and antagonist binding in receptors for neurotransmitters. These arginines were mutated and the mutant receptors were transiently expressed in COS-1 cells. The affinity of the R306K TRH-R was similar to that of the wild-type (WT) TRH-R, whereas no specific binding was detected in cells expressing R306A, R306E, or R306L TRH-Rs. Because TRH stimulated inositol phosphate (IP) formation to similar maximal extents in cells expressing WT and Arg-306 mutant TRH-Rs, relative potencies were used to estimate the relative affinities of the receptors. The EC50 values for stimulation of R306A, R306E, and R306L TRH-Rs were 1500-, 1200-, and 3000-fold higher than that for the WT TRH-R. No specific binding was measurable in COS-1 cells expressing R283K, R283H, or R283A TRH-Rs, whereas maximal TRH stimulation of IP formation was to levels 64%, 42%, or < 1%, respectively, of that in cells expressing WT TRH-Rs; for R283K and R283H TRH-Rs, EC50 values were 6300- and 50,000-fold higher, respectively, than that for the WT TRH-R. In AtT-20 cells stably expressing R283A TRH-Rs, the binding affinity was 39,000-fold lower than that of the WT TRH-R and the number of receptors was estimated to be 0.88 x 10(6)/cell, but TRH did not stimulate IP formation. Thus, in the TRH-R, Arg-306 appears to be important for binding but not for activation, whereas Arg-283 appears to be important for binding and activation.
Mol Pharmacol 1995 Mar
PMID:Distinct roles for arginines in transmembrane helices 6 and 7 of the thyrotropin-releasing hormone receptor. 770 Feb 46

Familial male precocious puberty (FMPP) is a gonadotropin-independent disorder that is inherited in an autosomal dominant, male-limited pattern. A heterozygous mutation encoding substitution of Asp578 with Gly in transmembrane helix 6 of the G protein-coupled receptor for luteinizing hormone (LHR) has been found in affected males from nine American FMPP families. Cells expressing the mutant LHR exhibit markedly increased cyclic adenosine monophosphate (cAMP) production in the absence of agonist, suggesting that autonomous Leydig cell activity in FMPP is caused by a constitutively activated LHR. We have now analyzed genomic DNA from affected males from six additional FMPP families. PCR was used to amplify a fragment of the LHR gene encoding amino acid residues 441-594. None of the six new samples contained the Asp578-->Gly mutation, as indicated by absence of digestion with MspI. PCR products were then screened for heterozygous mutations using temperature-gradient gel electrophoresis. DNA fragments from two of the patients migrated abnormally. Direct sequencing of PCR product from one affected German male revealed a heterozygous mutation (ATG-->ATA) encoding Met571-->Ile at the cytoplasmic end of helix 6, the same mutation that has been reported in another European FMPP kindred. Affected males in the second family had a novel Thr577-->Ile mutation (ACC-->ATC). Mutations in different portions of the LHR or in a different gene may be responsible for disease in the other FMPP kindreds. Agonist binding and functional coupling of the mutant receptors to the cAMP and inositol phosphate pathways were studied by transiently expressing them in COS-7 cells. Agonist affinity was unaffected by the mutations.(ABSTRACT TRUNCATED AT 250 WORDS)
Hum Mol Genet 1995 Feb
PMID:Characterization of heterogeneous mutations causing constitutive activation of the luteinizing hormone receptor in familial male precocious puberty. 775 65

Gonadotropin-releasing hormone (GnRH) interacts with a G protein-coupled receptor and increases the transcription of the glycoprotein hormone alpha-subunit gene. We have explored the possibility that mitogen-activated protein kinase (MAPK) plays a role in mediating GnRH effects on transcription. Activation of the MAPK cascade by an expression vector for a constitutively active form of the Raf-1 kinase led to stimulation of the alpha-subunit promoter in a concentration-dependent manner. GnRH treatment was found to increase the phosphorylation of tyrosine residues of MAPK and to increase MAPK activity, as determined by an immune complex kinase assay. A reporter gene assay using the MAPK-responsive, carboxy-terminal domain of the Elk1 transcription factor was also consistent with GnRH-induced activation of MAPK. Interference with the MAPK pathway by expression vectors for kinase-defective MAPKs or vectors encoding MAPK phosphatases reduced the transcription-stimulating effects of GnRH. The DNA sequences which are required for responses to GnRH include an Ets factor-binding site. An expression vector for a dominant negative form of Ets-2 was able to reduce GnRH effects on expression of the alpha-subunit gene. These findings provide evidence that GnRH treatment leads to activation of the MAPK cascade in gonadotropes and that activation of MAPK contributes to stimulation of the alpha-subunit promoter. It is likely that an Ets factor serves as a downstream transcriptional effector of MAPK in this system.
Mol Cell Biol 1995 Jul
PMID:A role for mitogen-activated protein kinase in mediating activation of the glycoprotein hormone alpha-subunit promoter by gonadotropin-releasing hormone. 779 60

Three full-length cDNA clones encoding the guinea pig A1 adenosine receptor have been isolated by polymerase chain reaction (PCR) and low-stringency hybridization screening of a guinea pig brain cDNA library. These three cDNAs, though differing in their 5' untranslated regions, contain the same open reading frame encoding a 326 amino acid residue protein with seven hydrophobic alpha-helices long enough to form the transmembrane domains, suggesting that it belongs to the G protein-coupled receptor superfamily. This protein is highly homologous to the A1 adenosine receptors previously cloned from other species. Pharmacological characterization of this receptor transiently expressed in mammalian cells demonstrates that, despite its high homology to A1 adenosine receptors of other species, the guinea pig A1 adenosine receptor displays a unique pharmacological profile: high affinity for the A1-selective antagonist CPX, yet very low affinity for some A1-selective agonists such as CCPA, CHA and R-PIA. Northern blotting for different guinea pig tissues and in situ hybridization for guinea pig brain sections reveal an abundant and broad distribution of mRNA of this A1 subtype receptor in the brain.
Brain Res Mol Brain Res 1994 Oct
PMID:Cloning and characterization of a pharmacologically distinct A1 adenosine receptor from guinea pig brain. 785 41

Agonist-dependent phosphorylation of G protein-coupled receptors (GPRs) by G protein-coupled receptor kinases (GRKs) is proposed to be a key event initiating homologous receptor desensitization. A technical limitation hindering identification of GPRs as GRK substrates has been the necessity to use purified and reconstituted receptors in GRK assays. Here, the human m2 and human m3 (hm3) muscarinic cholinergic receptors (mAChRs), which couple to attenuation of adenylyl cyclase and stimulation of phospholipase C, respectively, were expressed in Spodoptera frugiperda insect cells and an in vitro approach to studying GPR phosphorylation by GRKs in crude membranes was developed. The m2 mAChR, a known substrate of certain GRKs, was used to validate the approach. The GRK isoform beta-adrenergic receptor kinase (beta ARK)1 phosphorylated the membrane-bound human m2 mAChRs in an agonist-dependent manner. The results demonstrated that endogenous membrane-bound beta gamma subunits of G proteins stimulated the phosphorylation of the membrane-bound m2 mAChR. To reveal new GRK substrates, we tested the expressed hm3 mAChRs. The membrane-bound hm3 mAChRs were phosphorylated by beta ARK1 in an agonist-dependent, G beta gamma-enhanced manner. This is the first demonstration that hm3 mAChRs can serve as substrates for GRKs. The stoichiometry of receptor phosphorylation was approximately 2 mol of phosphate/mol of receptors in the absence of G beta gamma and approximately 4 mol of phosphate/mol of receptors upon addition of G beta gamma. When the specificity of various GRKs towards mAChRs was assessed, beta ARK2 phosphorylated the agonist-activated hm3 mAChRs as efficiently as did beta ARK1; however, neither GRK5 nor GRK6 significantly phosphorylated the hm3 mAChRs under similar conditions. The approach of studying GRK-mediated phosphorylation of GPRs in their membrane-bound state identified the hm3 mAChRs as new substrates for GRKs. This approach should be valuable in identifying other new substrates of GRKs and should aid in studies that elucidate GRK/GPR pairing.
Mol Pharmacol 1995 Feb
PMID:Agonist-dependent phosphorylation of human muscarinic receptors in Spodoptera frugiperda insect cell membranes by G protein-coupled receptor kinases. 787 29

Despite the importance of beta 2-adrenergic receptor (beta 2AR) stimulation in mediating airway epithelial cell function, little is known regarding its regulation in airway epithelium. Perturbations of the airway environment associated with disease states, including the management of bronchomotor tone with beta-agonists, expose airways to putative regulators of beta 2AR signal transduction. In this communication, we describe the desensitization of beta 2AR signal transduction in the human airway epithelial cell line BEAS-2B. Examination of both beta-agonist- and prostaglandin E2 (PGE2)-mediated cAMP generation in BEAS-2B cells reveals both agonist-specific (homologous) and non-agonist-specific (heterologous) desensitization of these G protein-coupled receptor pathways. Short-term homologous desensitization of beta 2AR-mediated cAMP generation was characterized by an approximately 60% loss of maximal responsiveness to isoproterenol (ISO) when cells were pretreated 30 min with 10 microM ISO. A reduced sensitivity to ISO was also evidenced by an approximately 4-fold increase in the EC50 for ISO stimulation of adenylyl cyclase (AC). Short-term heterologous desensitization was characterized by an increase in EC50 (approximately 2- to 3-fold) with no change in maximal responsiveness to ISO in cells pretreated with either forskolin or PGE2. Qualitatively similar findings characterized short-term homologous and heterologous desensitization of PGE2-mediated AC activity. Short-term agonist-specific desensitization of the beta 2AR was associated with, but not dependent upon, rapid beta 2AR sequestration. Long-term pretreatment of cells with 10 nM ISO and 1 microM PGE2 eliminated AC responsiveness to subsequent ISO and PGE2 stimulation, respectively. Exposure of BEAS-2B cells to ISO for 24 h resulted in an approximately 70% loss of beta 2ARs, whereas chronic forskolin or PGE2 pretreatment had no effect on beta 2AR number. Long-term pretreatment of cells designed to elicit heterologous desensitization was associated with reductions in maximal responsiveness to ISO and PGE2 that appear to be related to a loss in inherent AC activity. These findings hold strong implications regarding the effect of beta 2AR desensitization on epithelial cell function and the role of beta-agonists in the management of airway disease.
Am J Respir Cell Mol Biol 1994 Oct
PMID:Regulation of beta-agonist- and prostaglandin E2-mediated adenylyl cyclase activity in human airway epithelial cells. 791 18


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