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Query: UNIPROT:P06889 (Mol)
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Five separate guanine nucleotide-binding proteins (G proteins) were immunologically identified in membranes from neuroblastoma x glioma NG108-15 hybrid cells. These alpha subunit proteins were Gi2 alpha, two isoforms of Gi3 alpha, and two isoforms of Go alpha. The G proteins that interacted with delta-opioid receptors in these membranes were identified using cholera toxin (CTX)-induced ADP-ribosylation and antisera selective for various G protein alpha subunits. In the presence of delta-opioid agonists, CTX induced the incorporation of [32P]ADP-ribose into three pertussis toxin substrates. Using antisera generated against peptide sequences from G alpha subunits, these three pertussis toxin substrates were identified as Gi2 alpha, Go2 alpha, and one isoform of Gi3 alpha, which has yet to be identified. This CTX-induced labeling was demonstrated to be mediated via the delta-opioid receptor in these hybrid cells by the observation that delta agonists D-Ala2-D-Leu5-enkephalin (DA-DLE) and D-Pen2-D-Pen5-enkephalin, as well as the nonselective agonists etorphine and bremazocine, were active, but the mu agonist PL017 and the kappa agonist U-50-488H did not show this activity. This incorporation into all three substrates induced by DADLE was dose dependent, with EC50 (95% confidence interval) values ranging from 12 (3-52) to 183 (65-520) nM, which compared with the Kd value of 10 +/- 1.5 nM for this agonist, a dose that produces maximal inhibition of adenylate cyclase activity. Furthermore, pretreatment of the cells with pertussis toxin or treatment of the membranes with the antagonist naloxone blocked the incorporation induced by DADLE. Incorporation of [32P]ADP-ribose into all three substrates decreased 35-83% in membranes in which the receptors had been down-regulated by chronic treatment of the cells with DADLE. Thus, a single opioid receptor type can interact with three separate G proteins.
Mol Pharmacol 1992 May
PMID:Identification of three separate guanine nucleotide-binding proteins that interact with the delta-opioid receptor in NG108-15 neuroblastoma x glioma hybrid cells. 131

A 2.6 kb plasmid, named pBBR1, was isolated from Bordetella bronchiseptica S87. After insertion of an antibiotic resistance marker, this plasmid could be transferred into Escherichia coli, Bordetella pertussis, B. bronchiseptica, Vibrio cholerae, Rhizobium meliloti, and Pseudomonas putida by transformation or conjugation. Conjugation was possible only when the IncP group transfer functions were provided in trans. As shown by incompatibility testing, pBBR1 does not belong to the broad-host-range IncP, IncQ or IncW groups. DNA sequence analysis revealed two open reading frames: one was called Rep, involved in replication of the plasmid, and the other, called Mob, was involved in mobilization. Both the amino-terminal region of Mob and its promoter region show sequence similarities to Mob/Pre proteins from plasmids of Gram-positive bacteria. In spite of these sequence similarities, pBBR1 does not replicate via the rolling-circle mechanism commonly used by small Gram-positive plasmids. We therefore speculate that pBBR1 may combine a mobilization mechanism of Gram-positive organisms with a replication mechanism of Gram-negative organisms. Determination of the plasmid copy number in E. coli and B. pertussis indicated that pBBR1 has a rather high copy number, which, in conjunction with its small size and broad host range, renders it particularly interesting for studies of broad-host-range replicons and for the development of new cloning vectors for a wide range of Gram-negative bacteria.
Mol Microbiol 1992 Jul
PMID:Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms. 132 24

Recently, a defect in pertussis toxin-independent actions of epinephrine on pancreatic B-cells of fa/fa Zucker rats was reported (Cawthorn and Chan (1991) Mol. Cell. Endocrinol. 75, 197-204). We now report studies of islet alpha 2-adrenoceptor function of fa/fa rats. Insulin and cAMP production by islets of obese rats were both inhibited by the alpha 2-adrenoceptor agonist clonidine. Calculated pD2 values for clonidine were 9.57 +/- 0.59 and 9.43 +/- 0.33 for lean and fa/fa rat islets, respectively. Yohimbine reversed clonidine effects equipotently in lean and obese rat islets (pA2 values of 7.48 +/- 0.57 vs 7.43 +/- 0.58). Unexpectedly, the alpha 1-antagonist prazosin stimulated insulin secretion from islets of obese but not lean rats. Functional characteristics of the alpha-adrenoceptors on fa/fa islets are thus similar to those recently designated alpha 2B. Altered expression of alpha-adrenoceptors on pancreatic islets of fa/fa rats may contribute to changes in the pertussis toxin-independent pathway of epinephrine action previously observed.
Mol Cell Endocrinol 1992 Mar
PMID:Functional characterization of alpha-adrenoceptors on pancreatic islets of fa/fa Zucker rats. 132 30

NIH-3T3 fibroblasts have been transfected with human serotonin 5-HT1A receptors. Clonal cell lines expressed between 40 and 500 fmol receptor/mg. 5-HT1A agonists strongly inhibited nonstimulated- as well as forskolin- or isoproterenol-stimulated adenylyl cyclase. The effects of 5-HT1A receptor activation on cell growth were investigated. 5-HT1A agonists accelerated cell division, generated foci, and increased DNA synthesis. The stimulation of [3H]thymidine incorporation was much stronger when tyrosine kinase receptors were activated concomitantly. Cyclic AMP (cAMP) elevating agents inhibited DNA synthesis induced by all mitogens tested. The mitogenic activity of 5-HT1A agonists did not seem to be linked to adenylyl cyclase inhibition because 1) we were not able to measure any decrease in intracellular cAMP levels under the conditions of DNA synthesis assay and 2) 2',5'-dideoxyadenosine, which strongly inhibited adenylyl cyclase, was not mitogenic and did not modify the mitogenic effects of 5-HT1A agonists. Pertussis toxin completely blocked potentiation of epidermal growth factor effect induced by 8-hydroxy-di-(n-propyl)aminotetralin, a 5-HT1A agonist, but only partially blocked the one induced by insulin. In conclusion, in transfected NIH-3T3 cells, transforming and mitogenic effects of 5-HT1A agonists involve a pertussis toxin-sensitive G protein but do not seem to be linked to adenylyl cyclase inhibition.
Mol Biol Cell 1992 Sep
PMID:Activation of 5-HT1A receptors expressed in NIH-3T3 cells induces focus formation and potentiates EGF effect on DNA synthesis. 133 92

The rat neurotensin receptor cDNA sequence was transfected in Chinese hamster ovary cells and cellular clones which stably express the corresponding protein were isolated and characterized. The Scatchard analysis of the specific binding of [3H]neurotensin indicated a Kd value of 0.45 +/- 0.08 nM and a Bmax value of 3.27 +/- 0.29 pmol/mg of protein. Displacement experiments using peptidic analogs of neurotensin and levocabastine confirmed that the transfected receptor exhibits the binding properties of the neurotensin receptor characterized in the rat brain. Neurotensin stimulated the phosphoinositides hydrolysis in a time- and concentration-dependent manner and this effect was mimicked by neurotensin(8-13) and by neuromedin N. The stimulation of phosphoinositides hydrolysis was not inhibited by pertussis toxin. These results indicate that the transfected cells actively express the rat neurotensin receptor which is functionally coupled to phospholipase C through a pertussis toxin-insensitive GTP-binding protein, and that neuromedin N is able to induce the phosphoinositides turnover by interaction with the neurotensin receptor.
Brain Res Mol Brain Res 1992 Oct
PMID:Phospholipase C activation by neurotensin and neuromedin N in Chinese hamster ovary cells expressing the rat neurotensin receptor. 133 89

Chronic stimulation of beta-adrenoceptors leads to increased mRNA and protein levels of pertussis toxin (PTX)-sensitive guanine nucleotide-binding proteins (G proteins) in the heart. In the present study the time course is reported of the effect of isoprenaline infusions on myocardial mRNA levels of Gi alpha-2, Gi alpha-3, G(o) alpha, Gs alpha, and G beta and myocardial levels of PTX-sensitive G proteins. Rats were treated by subcutaneous infusions, with osmotic minipumps, of 0.9% NaCl, isoprenaline (2.4 mg/kg/day), propranolol (9.9 mg/kg/day), or a combination thereof for 1, 2, 3, 4, or 8 days, and two groups were treated with NaCl or isoprenaline for 13 or 26 days. Additional groups of rats were treated with 0.24 or 0.07 mg/kg/day for 4 days to determine the dose dependency of the effects of isoprenaline. mRNA concentrations were determined by standardized slot blotting with 32P-labeled cDNA or cRNA probes. In isoprenaline-treated rats, mRNA levels of all members of the Gi alpha/G(o) alpha family increased gradually in parallel. The increase in Gi alpha-2, Gi alpha-3, and G(o) alpha mRNA levels reached significance on days 2-3, reached maximal values on days 3-4, and remained stable over the total time of treatment of up to 26 days. Compared with NaCl, maximal increases were 77% (Gi alpha-2; 16.4 versus 9.3 pg/micrograms), 58% (Gi alpha-3; 4.65 versus 2.95 pg/micrograms), and 78% (G(o) alpha; 0.40 versus 0.22 pg/microgram). Gs alpha mRNA levels (about 30 pg/micrograms) and G beta mRNA levels remained unchanged. In isoprenaline-treated rats myocardial levels of PTX-sensitive G proteins increased by maximally 54%, compared with control. The time course differed slightly from the time course of mRNA up-regulation in the first 3 days of treatment and paralleled the increase in mRNA levels from day 4 on. Propranolol had no effect on G alpha mRNA or protein levels when given alone but abolished all effects of isoprenaline when given in combination. Isoprenaline at a dose of 0.24 mg/kg/day still induced an increase in Gi alpha-2 mRNA levels of 24%, without any effects on histopathology of the myocardium.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1992 Nov
PMID:Long term beta-adrenoceptor-mediated up-regulation of Gi alpha and G(o) alpha mRNA levels and pertussis toxin-sensitive guanine nucleotide-binding proteins in rat heart. 133 60

The mechanism(s) underlying adenosine receptor-mediated modulation of cardiac cAMP levels has been investigated using detergent-permeabilized embryonic chick ventricular myocytes. The beta-adrenergic receptor agonist isoproterenol (ISO) stimulated adenylyl cyclase activity in detergent-permeabilized cells by 5-10-fold, with an EC50 value of 0.3 microM. Three adenosine receptor agonists, (R)-N6-phenylisopropyladenosine, N6-(3-iodo-4-aminobenzyl)adenosine, and 5'-N-ethylcarboxamidoadenosine, inhibited ISO (10 microM)-stimulated adenylyl cyclase activity in a concentration-dependent manner. The maximum inhibition of the ISO-stimulated adenylyl cyclase activity by (R)-N6-phenylisopropyladenosine (10 microM) was 30-40%. This inhibition was antagonized by the adenosine receptor antagonists xanthine amine congener and 8-cyclopentyl-1,3-dipropylxanthine and was abolished by pertussis toxin treatment, suggesting that the inhibition of adenylyl cyclase activity is mediated by A1 adenosine receptors acting via a pertussis toxin-sensitive guanine nucleotide-binding protein (G protein). Because the adenosine receptor agonists had no detectable effect on phosphodiesterase activity, the adenosine receptor-mediated inhibition of adenylyl cyclase activity appears to account for the cAMP-lowering effect of adenosine receptor agonists seen in intact cardiac myocytes. Moreover, two A1 adenosine receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine and 3-(4-amino)phenethyl-1-propyl-8-cyclopentylxanthine, stimulated basal adenylyl cyclase activity in the absence of an adenosine receptor agonist; this stimulation was abolished by pretreatment of the cells with pertussis toxin. We postulate that "precoupled" A1 adenosine receptor-G protein complexes, present in the cardiac myocytes, exert a tonic inhibitory influence on adenylyl cyclase activity and that some adenosine receptor antagonists remove this tonic inhibition by destabilizing these precoupled receptor-G protein complexes.
Mol Pharmacol 1992 Nov
PMID:Modulation of cardiac cyclic AMP metabolism by adenosine receptor agonists and antagonists. 133 65

The regulation of pituitary hormone secretion by TRH and GnRH proceeds through similar mechanisms which employ phosphoinositide hydrolysis to generate intracellular signals. Proximal events involve receptor activation of heterotrimeric (alpha beta gamma) GTP-binding (G) proteins which regulate phospholipase (PLC) activity. Since TRH and GnRH actions are not affected by cholera or pertussis toxin, a novel G protein (Gp) was suggested to mediate receptor regulation. The required Gp protein has not been identified and this was the focus of the present study. Recent molecular cloning and biochemical studies have characterized two novel, pertussis toxin-insensitive alpha-subunit proteins of the Gq subfamily (alpha q and alpha 11) which regulate the activity of the beta 1 isoenzyme of PLC. Gq and G11 represent the best candidates for the PLC-activating G proteins which mediate the actions of TRH and GnRH. To test this directly, an antibody to the common Gq/11 alpha-subunit carboxyterminal sequence was generated and shown to react with unique 42-kilodalton Gq alpha and 43-kilodalton G11 alpha proteins in membranes from TRH-responsive GH3 cells and GnRH-responsive alpha T3-1 pituitary cells. The Gq/11 alpha peptide antibody was shown to immunodeplete the Gp activity of GH3 cell membrane extracts measured by reconstitution of the guanine nucleotide regulation of PLC-beta 1. In addition, the immunoglobulin G fraction of Gq/11 alpha peptide immune serum specifically inhibited TRH- and GnRH-stimulated PLC activity measured in the membranes of GH3 and alpha T3-1 cells, respectively. The results indicate that TRH and GnRH activation of PLC requires receptor coupling to a Gp protein(s) which corresponds to Gq, G11 or both.
Mol Endocrinol 1992 Oct
PMID:Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to the guanosine triphosphate-binding proteins Gq and G11. 133 52

The high selectivity, low conductance, amiloride-blockable, sodium channel of the mammalian distal nephron (i.e. cortical collecting tubule) is the site of discretionary regulation which allows maintainance of total body sodium balance. In order to understand the physiological events that participate in this regulation, we have used the patch-clamp technique which allows us to measure individual Na+ channel currents and permits access to the cytosolic side of the channel-protein as well as its associated regulatory components. Most of our experiments have utilized the A6 amphibian renal cell line, which when grown on permeable supports is an excellent model for the mammalian distal nephron. Different mechanisms have been examined: (1) regulation by hormonal factors such as Anti-Diuretic Hormone (ADH) and aldosterone, (2) regulation by G-proteins, (3) modulation by protein kinase C (PK-C), and (4) modulation by products of arachidonic acid metabolism. Consistent with noise analysis of tight epithelial tissues, ADH treatment increased the number of active channels in apical membrane patches of A6 cells, without any apparent change in the open probability (Po) of the individual channels. Agents that increased intracellular cAMP mimicked the effects of ADH. In contrast, aldosterone was found to act through a dramatic increase in Po rather than through changes in channel density. Inhibition of methylation by deazaadenosine antagonizes the stimulatory effect of aldosterone. In excised inside-out patches GTP gamma S inhibits channel activity, whereas GDP beta S or pertussis toxin stimulates activity suggesting regulatory control by G-proteins. PK-C has been shown to contribute to 'feed-back inhibition' of apical Na+ conductance in tight epithelia.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1992 Sep 08
PMID:Regulation of renal epithelial sodium channels. 133 27

Somatostatin (SRIF) receptors are coupled to the catalytic subunit of adenylyl cyclase via pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins). To identify which G proteins link SRIF receptors to adenylyl cyclase, G(o) alpha, Gi alpha, and its different subtypes were individually blocked in AtT-20 cell membranes with G alpha subtype-selective antisera. Antiserum directed against the carboxyl-terminal region of Gi alpha blocked SRIF inhibition of forskolin-stimulated adenylyl cyclase activity, and this effect was prevented by the peptide to which the antiserum was generated. However, antiserum directed against the carboxyl-terminal region of G(o) alpha did not affect SRIF inhibition of adenylyl cyclase activity, indicating that Gi alpha couples SRIF receptors to adenylyl cyclase but G(o) alpha does not. Peptide-directed antisera against Gi alpha 1 completely blocked SRIF inhibition of adenylyl cyclase activity. In contrast, antisera directed against either Gi alpha 2 or Gi alpha 3 did not affect the actions of SRIF. The results of these studies indicate that Gi alpha 1 selectively couples SRIF receptors to the catalytic subunit of adenylyl cyclase in AtT-20 cell membranes. Because previous studies have shown that SRIF receptors are able to couple to Gi alpha 1, Gi alpha 3, and G(o) alpha, the results suggest that different G proteins may specify the coupling of SRIF receptors to distinct cellular effector systems.
Mol Pharmacol 1992 Mar
PMID:Gi alpha 1 selectively couples somatostatin receptors to adenylyl cyclase in pituitary-derived AtT-20 cells. 134 39


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