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Query: UNIPROT:P06889 (Mol)
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Using the polymerase chain reaction technique with degenerative primers, we obtained from a rat pituitary cDNA library a cDNA fragment, rAP236, that exhibited considerable homology to known receptors that belong to the guanine nucleotide-binding protein (G protein)-coupled receptor superfamily. Oligonucleotides to this fragment were used as probes to obtain a full-length cDNA from the rat pituitary cDNA library. This clone, rAP6-26, encoded a 383-amino acid protein with seven putative transmembrane domains that are characteristic of G protein-coupled receptors. The predicted amino acid sequence of the rAP6-26 cDNA exhibits 56-66% homology to recently cloned somatostatin (SRIF) receptors. Membranes prepared from COS-7 cells transfected with the rAP6-26 cDNA showed specific binding of 125I-Tyr11-SRIF, thus identifying the cDNA clone as a novel SRIF receptor. Radioligand binding competition analysis using somatostatin-28 (SRIF-28) and a number of cyclic SRIF analogs revealed that SRIF-28 was the most potent competitor of 125I-Tyr11-SRIF binding, with a approximately 30-fold greater affinity for the receptor than that of SRIF. In addition, binding of 125I-Tyr11-SRIF was markedly reduced in the presence of Na+ ions and GTP, indicating coupling of rAP6-26 receptors to inhibitory G proteins in COS-7 membranes. In adenylyl cyclase assays, forskolin-induced cAMP accumulation was inhibited by SRIF and SRIF-28, thus confirming that the rAP6-26 cDNA encodes a functional receptor protein. By Northern blot analysis, a approximately 2.6 kilobase mRNA encoding the receptor was present in the pituitary but not in the liver, small intestine, kidney, pancreas, cerebellum, or cortex. Lack of receptor mRNA expression in the brain was confirmed by in situ hybridization histochemical studies. Thus, we report the cloning of a novel rat pituitary SRIF receptor, termed SSTR4, that has marked preferential affinity for SRIF-28 and is linked to inhibition of adenylyl cyclase.
Mol Pharmacol 1992 Dec
PMID:Molecular cloning and expression of a pituitary somatostatin receptor with preferential affinity for somatostatin-28. 826 65

In the yeast Saccharomyces cerevisiae, adenylyl cyclase is regulated by RAS proteins. We show here that the yeast adenylyl cyclase forms at least two high-molecular-weight complexes, one with the RAS protein-dependent adenylyl cyclase activity and the other with the Mn(2+)-dependent activity, which are separable by their size difference. The 70-kDa adenylyl cyclase-associated protein (CAP) existed in the former complex but not in the latter. Missense mutations in conserved motifs of the leucine-rich repeats of the catalytic subunit of adenylyl cyclase abolished the RAS-dependent activity, which was accompanied by formation of a very high molecular weight complex having the Mn(2+)-dependent activity. Contrary to previous results, disruption of the gene encoding CAP did not alter the extent of RAS protein-dependent activation of adenylyl cyclase, while a concomitant decrease in the size of the RAS-responsive complex was observed. These results indicate that CAP is not essential for interaction of the yeast adenylyl cyclase with RAS proteins even though it is an inherent component of the RAS-responsive adenylyl cyclase complex.
Mol Cell Biol 1992 Nov
PMID:The 70-kilodalton adenylyl cyclase-associated protein is not essential for interaction of Saccharomyces cerevisiae adenylyl cyclase with RAS proteins. 140 71

The adenylyl cyclases of both Saccharomyces cerevisiae and Schizosaccharomyces pombe are associated with related proteins named CAP. In S. cerevisiae, CAP is required for cellular responses mediated by the RAS/cyclic AMP pathway. Both yeast CAPs appear to be bifunctional proteins: the N-terminal domains are required for the proper function of adenylyl cyclase, while loss of the C-terminal domains results in morphological and nutritional defects that appear to be unrelated to the cAMP pathways. Expression of either yeast CAP in the heterologous yeast suppresses phenotypes associated with loss of the C-terminal domain of the endogenous CAP but does not suppress loss of the N-terminal domain. On the basis of the homology between the two yeast CAP proteins, we have designed degenerate oligonucleotides that we used to detect, by the polymerase chain reaction method, a human cDNA fragment encoding a CAP-related peptide. Using the polymerase chain reaction fragment as a probe, we isolated a human cDNA clone encoding a 475-amino-acid protein that is homologous to the yeast CAP proteins. Expression of the human CAP protein in S. cerevisiae suppresses the phenotypes associated with loss of the C-terminal domain of CAP but does not suppress phenotypes associated with loss of the N-terminal domain. Thus, CAP proteins have been structurally and, to some extent, functionally conserved in evolution between yeasts and mammals.
Mol Cell Biol 1992 Nov
PMID:Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins. 140 78

Using an expression cloning assay, we have isolated a novel cDNA, referred to as rsp-1, which suppresses the v-Ras-transformed phenotype. When introduced into NIH 3T3 fibroblasts under the control of a metallothionein promoter, rsp-1 confers resistance to v-Ras, but not to v-Mos or v-Src, and inhibits growth of the cells. The rsp-1 cDNA contains a 831-bp open reading frame encoding a 277-amino-acid leucine-rich protein. The rsp-1 cDNA exhibits no significant homology to sequences in the DNA data bases. However, searches of the protein data bases revealed that it contains a series of leucine-based repeats which are homologous to the leucine repeats found in the regulatory region of the yeast adenylyl cyclase. rsp-1 specific RNA is detectable in a wide variety of cell lines and tissues, and the gene is conserved among eukaryotic species. These data suggest that rsp-1 plays a role in Ras signal transduction.
Mol Cell Biol 1992 Sep
PMID:Isolation of rsp-1, a novel cDNA capable of suppressing v-Ras transformation. 150 80

7-(2-Aminoethyl)aminocarbonyl-7-desacetylforskolin (7-AEC-Fsk) and 6-(2-aminoethyl)aminocarbonylforskolin (6-AEC-Fsk) were synthesized and tested for their ability to activate adenylyl cyclase and inhibit the high affinity binding of [3H]forskolin to bovine brain membranes. Forskolin and 7-AEC-Fsk were equipotent in activating adenylyl cyclase, with EC50 values of about 4 microM, whereas 6-AEC-Fsk had an EC50 of about 2 microM. 6-AEC-Fsk and 7-AEC-Fsk stimulated adenylyl cyclase about 7-fold over basal levels at 100 microM, whereas forskolin produced a 5-fold stimulation. Forskolin and 6-AEC-Fsk inhibited the binding of [3H]forskolin to bovine brain membranes with Kd values of 41 nM and 28 nM, respectively, whereas 7-AEC-Fsk had a Kd of 83 nM. The 3-(3-iodo-4-hydroxyphenyl)propionamide derivative of 6-AEC-Fsk (6-I-HPP-Fsk) was more potent than forskolin in inhibiting [3H]forskolin binding to bovine brain membranes, with a Kd of 14 nM. 6-AEC-Fsk was reacted with 125I-labeled Bolton-Hunter reagent to produce 6-125I-HPP-Fsk with a specific activity of 2175 Ci/mmol. 6-125I-HPP-Fsk bound to bovine brain membranes with a Kd of 13 nM and a Bmax of 3.8 pmol/mg of protein. Forskolin inhibited the binding of 6-125I-HPP-Fsk to bovine brain membranes with a Kd of 31 nM, whereas 1,9-dideoxyforskolin only slightly inhibited the binding at 10 microM. The binding of 6-125I-HPP-Fsk was not inhibited by agents that inhibit forskolin binding to the glucose transporter, such as D-glucose or cytochalasin B. There was no displaceable binding of 6-125I-HPP-Fsk to red blood cell membranes, which contain a large concentration of the glucose transporter. Pretreatment of bovine brain membranes with an alkylating derivative of forskolin, 7-bromoacetyl-7-desacetylforskolin (BrAcFsk), led to an irreversible decrease in the binding of [3H]forskolin and 6-125I-HPP-Fsk. The time dependence and concentration dependence for the BrAcFsk-induced decrease in [3H]forskolin binding sites were identical to those observed for the decrease in 6-125I-HPP-Fsk binding sites. 6-125I-HPP-Fsk binding was determined in human platelet membranes in the presence of Mg2+ alone and in combination with guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or AIF4-. The presence of GTP gamma S or AIF4- increased the binding of 6-125I-HPP-Fsk by 4.5-fold and 4-fold, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1992 Feb
PMID:Interaction of aminoalkylcarbamates of forskolin with adenylyl cyclase: synthesis of an iodinated derivative of forskolin with high affinity for adenylyl cyclase. 153 12

We have identified, cloned, and studied a gene, cap, encoding a protein that is associated with adenylyl cyclase in the fission yeast Schizosaccharomyces pombe. This protein shares significant sequence homology with the adenylyl cyclase-associated CAP protein in the yeast Saccharomyces cerevisiae. CAP is a bifunctional protein; the N-terminal domain appears to be involved in cellular responsiveness to RAS, whereas loss of the C-terminal portion is associated with morphological and nutritional defects. S. pombe cap can suppress phenotypes associated with deletion of the C-terminal CAP domain in S. cerevisiae but does not suppress phenotypes associated with deletion of the N-terminal domain. Analysis of cap disruptants also mapped the function of cap to two domains. The functional loss of the C-terminal region of S. pombe cap results in abnormal cellular morphology, slow growth, and failure to grow at 37 degrees C. Increases in mating and sporulation were observed when the entire gene was disrupted. Overproduction of both cap and adenylyl cyclase results in highly elongated large cells that are sterile and have measurably higher levels of adenylyl cyclase activity. Our results indicate that cap is required for the proper function of S. pombe adenylyl cyclase but that the C-terminal domain of cap has other functions that are shared with the C-terminal domain of S. cerevisiae CAP.
Mol Biol Cell 1992 Feb
PMID:Genetic and biochemical analysis of the adenylyl cyclase-associated protein, cap, in Schizosaccharomyces pombe. 155 Sep 59

A novel adenosine receptor subtype has been cloned from a rat brain cDNA library using a probe generated by the polymerase chain reaction. The cDNA, designated RFL9, encodes a protein of 332 amino acids. The structure of RFL9 is most similar to that of the recently cloned rat A2-adenosine receptor, with a sequence identity of 73% within the presumed seven transmembrane domains. Expression of RFL9 in COS-6M cells resulted in ligand binding and functional activity characteristics of an adenosine receptor that is coupled positively to adenylyl cyclase. Examination of the tissue distribution of RFL9 mRNA by Northern blot analysis showed a restricted distribution with highest levels expressed in large intestine, cecum, and urinary bladder; this pattern was distinct from that of either the A1- or A2-adenosine receptor mRNAs. In situ hybridization studies of RFL9 mRNA showed no specific hybridization pattern in brain, but a hybridization signal was readily observed in the hypophyseal pars tuberalis. Thus, RFL9 encodes a novel A2-adenosine receptor subtype.
Mol Endocrinol 1992 Mar
PMID:Molecular cloning and expression of the cDNA for a novel A2-adenosine receptor subtype. 158 14

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein.
Mol Cell Biol 1992 Jun
PMID:Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein. 158 63

Human serotonin [5-hydroxytryptamine (5-HT)1A] receptors have been transfected in NIH-3T3 cells, and their pharmacology and coupling to adenylyl cyclase have been analyzed. Three cellular preparations were used, 1) monoclonal cell lines (clones 6, 2B, and 4B), expressing 45, 280, and 500 fmol of 5-HT1A receptors/mg of protein, respectively; 2) clones 6, 2B, and 4B in which the concentration of 5-HT1A receptors was increased after stimulation of the glucocorticoid-inducible promoter with dexamethasone; and 3) polyclonal cell lines that expressed an increasing amount of 5-HT1A receptor as a function of cell passage. The transfected 5-HT1A receptors inhibited basal, forskolin-stimulated, and isoproterenol-stimulated adenylyl cyclase. The inhibition was dependent on the receptor density expressed, increasing from 60% at low density (45 fmol/mg) to 90% at a density higher than 280 fmol/mg. The pharmacology of the 5-HT1A receptor was studied, with particular attention being paid to the behavior of some agonists. These pharmacological characteristics are similar to those of 5-HT1A receptors in hippocampus but different from those of 5-HT1A in cerebral cortex. Analysis of the potencies and efficacies of the full agonist 5-HT and the partial agonist ipsapirone, as a function of receptor density in the three cellular populations used, revealed that 1) the efficacies of the full and partial agonists increased with the receptor density; 2) the EC50 values of the full and partial agonists were not shifted to the left when the receptor density was increased (based on the increase in efficacy and considering the classical pharmacological models of receptor-drug action, a 9-10-fold shift was expected); and 3) the ratio between the efficacies of the full agonist 5-HT and the partial agonist ipsapirone was not modified when the receptor concentration was increased or when the GTP-binding protein availability was decreased. The results indicate that neither the classical nor the operational model of drug-receptor action can be used to describe the coupling of 5-HT1A receptors to adenylyl cyclase in transfected NIH-3T3 cells. One of the explanations could be that 5-HT1A receptors and GTP-binding proteins are coupled in functional domains (almost precoupled), rather than distributed in homogeneous compartments in which they are free to diffuse.
Mol Pharmacol 1992 Jun
PMID:Transfection of human 5-hydroxytryptamine1A receptors in NIH-3T3 fibroblasts: effects of increasing receptor density on the coupling of 5-hydroxytryptamine1A receptors to adenylyl cyclase. 161 16

Acute myocardial ischaemia frequently is complicated by ventricular tachyarrhythmias. These arrhythmias are in part due to an increased susceptibility of myocardial cells to adenylyl cyclase stimulation by catecholamines [1]. As adenylyl cyclase underlies an endogenous dual regulation by stimulatory and inhibitory receptor systems, adenylyl cyclase stimulation can be counteracted by the activation of receptors like the muscarinic M2 receptor [2]. Therefore, the effect of myocardial ischaemia on muscarinic receptor and "inhibitory" guanine nucleotide binding proteins (G(i)) mediated inhibition of adenylyl cyclase was studied. During 5 min of myocardial ischaemia, carbachol mediated inhibition of forskolin and isoproterenol stimulated adenylyl cyclase was reduced by 30% and 50%, respectively. Hormone independent inhibition of adenylyl cyclase by the nonhydrolyzable GTP-analogue guanosine 5'-[beta gamma-imido]triphosphate (Gpp(NH)p) was reduced by 46%. In contrast, the amount of G(i), as determined by pertussis toxin catalyzed ADP-ribosylation, remained constant during 15 min of ischaemia. The impaired function of muscarinic receptor linked signal transduction during early myocardial ischaemia could contribute to the occurrence of ischaemia induced tachyarrhythmias by a reduced ability to counteract adenylyl cyclase activation.
J Mol Cell Cardiol 1992 May
PMID:Reduced adenylyl cyclase inhibition by carbachol and GTP during acute myocardial ischaemia. 163 72


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