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)

In this study, the influence of the inhibitory mu-opioid receptor on the potencies of 5'-guanosine alpha-thiotriphosphate (GTP gamma S) and GDP at the inhibitory GTP-binding protein (Gi) were investigated in an adenylyl cyclase system. It was hoped that a receptor-mediated change in the potency of either GTP gamma S or GDP in affecting adenylyl cyclase activity may elucidate how a receptor alters cyclase activity via its G-protein. In an adenylyl cyclase system employing 5'-adenylyl imidodiphosphate as substrate, GTP gamma S, a nonhydrolyzable analog of GTP, inhibited forskolin-stimulated adenylyl cyclase activity in the absence of morphine; morphine failed to significantly affect the apparent potency of GTP gamma S. GDP blocked the GTP gamma S-induced inhibition of adenylyl cyclase; morphine profoundly diminished the ability of GDP to block the inhibitory effect of GTP gamma S. The IC50 values of GTP gamma S were 0.02 +/- 0.01, 0.18 +/- 0.04, and 2.2 +/- 0.5 microM in the absence of other drugs, in the presence of a combination of 100 microM GDP and morphine, and in the presence of 100 microM GDP, respectively. GDP blocked the inhibitory effect of GTP gamma S (0.3 microM) in a concentration-dependent manner; the EC50 for GDP was 16 +/- 2.6 microM in the absence of morphine and 170 +/- 32 microM in the presence of morphine. Exposure of 7315c cells to pertussis toxin for 3 h resulted in a small decrease in the potency of GTP gamma S in inhibiting cyclase. However, the relative potency of GDP in blocking the GTP gamma S-mediated inhibition of cyclase was increased: the EC50 values of GDP were 11 +/- 4 and 0.81 +/- 0.2 microM in untreated and pertussis toxin-treated membranes, respectively. In untreated membranes, there was a brief lag in the GTP gamma S-induced inhibition of adenylyl cyclase; morphine diminished this lag. In membranes treated with pertussis toxin, there was an exaggerated lag in the onset of GTP gamma S inhibition of adenylyl cyclase activity; morphine could no longer affect this lag. Thus, uncoupling the mu-opioid receptor from Gi appeared to increase the affinity of Gi for GDP. These data suggest that the effect of an inhibitory receptor is to decrease the affinity of Gi for GDP by virtue of its interaction with the carboxy-terminal region of Gi alpha.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Endocrinol 1989 Feb
PMID:Site of pertussis toxin-induced ADP-ribosylation on Gi is critical for receptor modulation of GDP interaction with Gi. 254 Apr 29

When incubated at a restrictive temperature, Saccharomyces cerevisiae sec59 mutant cells accumulate inactive and incompletely glycosylated forms of secretory proteins. Three different secretory polypeptides (invertase, pro-alpha-factor, and pro-carboxypeptidase Y) accumulated within a membrane-bounded organelle, presumably the endoplasmic reticulum, and resisted proteolytic degradation unless the membrane was permeabilized with detergent. Molecular cloning and DNA sequence analysis of the SEC59 gene predicted an extremely hydrophobic protein product of 59 kilodaltons. This prediction was confirmed by reconstitution of the sec59 defect in vitro. The alpha-factor precursor, which was translated in a soluble fraction from wild-type cells, was translocated into, but inefficiently glycosylated within, membranes from sec59 mutant cells. Residual glycosylation activity of membranes of sec59 cells was thermolabile compared with the activity of wild-type membranes. Partial restoration of glycosylation was obtained in reactions that were supplemented with mannose or GDP-mannose, but not those supplemented with other sugar nucleotides. These results were consistent with a role for the Sec59 protein in the transfer of mannose to dolichol-linked oligosaccharide.
Mol Cell Biol 1989 Mar
PMID:Sec59 encodes a membrane protein required for core glycosylation in Saccharomyces cerevisiae. 265 87

Incubation of rat ovarian plasma membranes with [gamma-32P]guanosine 5'-triphosphate (GTP) in the presence of an adenosine triphosphate (ATP)-trapping system results in the labeling of a single protein, Mr 33,000 +/- 3000 designated 'a' (Amir-Zaltsman, Y., Ezra, E., Walker, M., Lindner, H. R. and Salomon, Y. (1980) FEBS Lett. 122, 166-170). Based on competition with other nucleotides it is concluded that protein 'a' is preferentially phosphorylated by [gamma-32P]GTP (Km = 0.28 microM). Phosphorylation of protein 'a' does not occur at pH less than 5 and progressively increases to plateau levels at pH 7-9. Phosphorylation of protein 'a' is absolutely dependent on the presence of divalent cations 1 mM Mg2+, Ca2+, or Cd2+. At higher concentrations, 5-20 mM, Mg2+ or in the presence of 1 mM Mn2+ ions other proteins are also phosphorylated. While vanadate ions selectively prevent the labeling of protein 'a', molybdate ions were found to inhibit phosphorylation of all the membrane proteins including protein 'a'. In contrast to molybdate ions, vanadate ions were found to accelerate the dephosphorylation of phosphoprotein 'a'. We suggest that phosphoprotein 'a' is a high energy protein intermediate in which the phosphate is present as a phosphoramidate for the following reasons: (i) Guanosine diphosphate (GDP) but not guanosine 5'-O-(2-thiodiphosphate) selectively accelerated the dephosphorylation of phosphoprotein 'a' but only in the presence of Mg2+ ions. (ii) The phosphoprotein intermediate is hydrolyzed in the presence of hydroxylamine. (iii) Phosphoprotein 'a' is labile in the presence of 1 N HCl but stable in 1 N NaOH at 37 degrees C. (iv) Phosphoprotein 'a' is heat labile. Phosphoprotein 'a' is readily digested by several proteolytic enzymes and a single cleavage peptide is generated upon treatment with Staphylococcus aureus V8 protease. The properties of protein 'a' were compared and found different from another phosphoprotein Mr 90,000 +/- 1000, designated 'b' that was selected arbitrarily. We propose that protein 'a' is a GTP requiring enzyme intermediate, of yet unidentified function.
Mol Cell Endocrinol 1989 May
PMID:Phosphorylation of proteins in rat ovarian plasma membranes by [gamma-32P]GTP: evidence for the formation of a high energy phosphoprotein. 275 26

An in vitro assay for phospholipase C activity was developed, employing exogenously added 32P-labelled phosphatidylinositol 4,5-bisphosphate as substrate. This enzymatic assay used to analyse the direct effect of GnRH on mammary tumors. GnRH agonists stimulate membranal phosphoinositide-specific phospholipase C activity. The increase in inositoltrisphosphate production is dose dependent, and is inhibited by the GnRH antagonist Org-30276. We took advantage of this non-cellular assay system for evaluating the role of G-binding proteins in the phosphoinositide transducing system in mammary tumors. GTP gamma S stimulates the basal and GnRH-dependent phospholipase C activity. This effect was abolished by GDP beta S. The cytosolic phospholipase C activity was also stimulated by GTP gamma S but was not affected by the hormone. These results suggest that GnRH may affect the growth of mammary tumors directly and not only through the reduction of blood gonadotropin level.
Mol Cell Endocrinol 1987 Oct
PMID:GnRH analogs stimulate phospholipase C activity in mammary tumor membranes: modulation by GTP. 282 15

Multiple affinity states of opioid receptors of the mu and delta types have been identified in membranes prepared from cells which bear only one type of opioid receptor (mu receptors in 7315c cells, delta receptors in NG 108-15 cells), and in guinea pig cortical membranes where both types of receptors were present in the membrane preparations. States of mu and delta receptors which have agonist affinities too low to be identified by radiolabeled agonist have been measured indirectly by agonist competition for sites labeled by radioactive antagonist. Using analogues of guanyl nucleotides, we have examined the competition of the mu and delta agonists DAGO and DSLET against [3H]DIP or [3H]NAL binding to opioid receptors and identified several agonist affinity states. In the absence of added nucleotide, competition of DSLET for [3H]DIP binding to delta opioid receptors revealed the presence of two binding sites with differing apparent agonist affinities. Addition of GDP beta S produced a steep monophasic curve which was best fit by a one-site model. In contrast, in the presence of added GTP or GTP gamma S, two affinity states were again apparent for DSLET competition at the delta receptor. The competition curve with GTP was shifted to the right relative to that produced in the absence of added guanyl nucleotide, indicating the presence of a lower apparent affinity state than any observed under other treatment conditions. DAGO competed against [3H]DIP or [3H]NAL binding to mu receptors over a wide concentration range in the absence of added guanyl nucleotide, consistent with the occupation by this ligand of more than one agonist affinity state of the mu receptor. However, when GDP beta S was added to the incubation mixture, only a single binding site was identified. Two mu receptor affinity states were again observed in the presence of added GTP or GTP gamma S. One of these had significantly lower apparent affinity than those states detected in the absence of added nucleotide or with GDP beta S. Pertussis toxin treatment resulted in a monophasic agonist competition curve which was best fitted by a single-site model in both 7315c and NG108-15 cell membranes. Addition of 100 microM GTP did not affect the agonist Kapp or Bmax after pertussis toxin treatment, suggesting that sites labeled under these conditions were not functionally associated with a G protein. In general, the effects of guanyl nucleotides were qualitatively similar at mu and delta receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1988 Apr
PMID:Multiple agonist-affinity states of opioid receptors: regulation of binding by guanyl nucleotides in guinea pig cortical, NG108-15, and 7315c cell membranes. 283 86

Guanine nucleotides have been examined as to their effects on subclass-specific excitatory amino acid receptor-ligand interactions. Guanine nucleotides selectively inhibit L-[3H]glutamate binding to the N-methyl-D-aspartate (NMDA) recognition site while showing a lesser effect on [3H]kainate, [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and sodium-dependent L-[3H]glutamate binding. Of the series of guanine nucleotides tested in the inhibition of NMDA-specific L-[3H]glutamate binding, GTP, GDP, 5'-guanylylimidodiphosphate and 5'-guanylylmethylenediphosphate were significantly more potent than GMP, cyclic GMP and guanosine. Scatchard analysis indicates that the GTP inhibition (IC50 = 28 microM) of this NMDA-specific L-[3H]glutamate binding results from a decrease in the affinity of L-glutamate for the NMDA receptor whereas no alteration in the number of binding sites is observed. A kinetic analysis indicates that this decrease in affinity may be attributed to a decrease in association rate whereas no change in dissociation rate is observed. GTP (25 microM) lowers the affinities of both NMDA agonists (NMDA, L-glutamate, L-aspartate, and L-homocysteate) and antagonists (D-2-amino-5-phosphonovalerate, D-2-amino-7-phosphonoheptanoate, and D-2-aminoadipate). Pretreatment of the synaptic plasma membranes with either pertussis or cholera toxin had no significant effect on the GTP inhibition of NMDA-specific L-[3H] glutamate binding. The data suggest that guanine nucleotides can negatively modulate the NMDA receptor; however, the mechanism of this modulation is unclear.
Mol Pharmacol 1988 Aug
PMID:Effects of guanine nucleotides on N-methyl-D-aspartate receptor-ligand interactions. 284 50

The effect of the new cardiotonic agent sulmazole on the guanine nucleotide regulatory protein-adenylate cyclase system was studied in rat adipocyte membranes. The inotrope enhanced basal adenylate cyclase activity by 46%. This stimulation occurred only at GTP concentrations (5 microM) sufficient to activate Gi. This stimulatory effect of sulmazole was abolished after functional inactivation of Gi, either by pertussis toxin or by using 10 nM GTP in the assay mixture, suggesting an important role of an active Gi in this process. Similarly, sulmazole enhanced isoproterenol-, forskolin-, and fluoride-stimulated adenylate cyclase activity by 33, 34, and 45%, respectively. However, when these latter experiments were performed after inactivation of Gi, sulmazole actually inhibited by approximately 25% adenylate cyclase activity stimulated by 1 and 10 microM isoproterenol. Under similar treatment conditions, enhancement of forskolin- and fluoride-stimulated activity by sulmazole was abolished. Sulmazole inhibited in a dose-dependent manner pertussis toxin- and cholera toxin-catalyzed labeling of Gi and Gs, respectively, with the respective inhibition observed at 100 microM of the inotrope being 29% and 56% of control. In addition, sulmazole inhibited PGE1 and isoproterenol-stimulated [3H]GDP release from Gi and Gs to 32% and 64% of control, respectively. Finally, the inotrope completely abolished PGE1-stimulated [3H]Gpp(NH)p binding with IC50 in the low micromolar range. These findings suggest that, whereas sulmazole inhibits the functioning of Gi and (to a lesser extent) Gs at low micromolar concentrations, expression of these effects on adenylate cyclase activity requires high micromolar to low millimolar concentrations of the drug. Thus, it appears sulmazole inhibits the function of Gi by decreasing its activation process, i.e., GTP-GDP exchange. Effects on Gs are manifested (at least in terms of adenylate cyclase activity) only after inactivation of Gi.
Mol Pharmacol 1988 Dec
PMID:The new positive inotrope sulmazole inhibits the function of guanine nucleotide regulatory proteins by affecting GTP turnover. 284 44

TRH receptors have been solubilized from GH4C1 cells using the plant glycoside digitonin. Solubilized receptors retain the principal binding characteristics exhibited by the TRH receptor in intact pituitary cells and their membranes. The binding of the methylhistidyl derivative of TRH [( 3H]MeTRH) attained equilibrium within 2-3 h at 4 C, and it was reversible, dissociating with a t1/2 of 7 h. Analysis of [3H]MeTRH binding to soluble receptors at 4 C yielded a dissociation constant (Kd) of 3.8 nM and a total binding capacity (Bmax) of 3.9 pmol/mg protein. Peptides known to interact with non-TRH receptors on GH cells failed to interfere with the binding of [3H]MeTRH, indicating that the TRH binding was specific. Chlordiazepoxide, a competitive antagonist for TRH action in GH cells, inhibited TRH binding to soluble receptors with an IC50 of 11 microM. When [3H]MeTRH was bound to membranes and the membrane proteins were then solubilized, we found enhanced dissociation of the prebound [3H]MeTRH from its solubilized receptor by guanyl nucleotides. Maximal enhancement of [3H]MeTRH dissociation by 10 microM GTP gamma S occurred within about 45 min at 22 C. GTP gamma S, GTP, GDP beta S, and GDP were all effectors of [3H]MeTRH dissociation, exhibiting EC50s in the range of 14-450 nM. The rank order of potency of the tested nucleotides was GTP gamma S greater than GTP congruent to GDP beta S greater than GDP much greater than ATP gamma S greater than GMP. We conclude that TRH receptors have been solubilized from GH cells with digitonin and retain the binding characteristics of TRH receptors in intact pituitary cells. Furthermore, prebinding [3H]MeTRH to GH4C1 cell membranes results in the solubilization of a complex in which the TRH receptor is linked functionally to a GTP binding protein.
Mol Endocrinol 1987 Dec
PMID:Solubilization of receptors for thyrotropin-releasing hormone from GH4C1 rat pituitary cells: demonstration of guanyl nucleotide sensitivity. 285 5

Activation of muscarinic cholinergic receptors of 1321N1 human astrocytoma cells attenuates cyclic AMP accumulation. This effect results from an activation of phosphodiesterase with no direct inhibition of adenylate cyclase activity. In spite of this lack of coupling of muscarinic receptors to adenylate cyclase, guanine nucleotides reduce the apparent binding affinity of the agonist carbachol in a washed membrane preparation of 1321N1 cells. The order of potency for this effect is guanosine 5'-O-(3-thiotriphosphate) greater than 5'-guanylyl-imidodiphosphate = GTP = GDP; ATP has no effect. The occurrence of a Mr = 41,000 protein labeled in the presence of [32P]NAD and pertussis toxin as well as the occurrence of guanine nucleotide-mediated inhibition of forskolin-stimulated adenylate cyclase activity indicate that the functional inhibitory guanine nucleotide regulatory component of adenylate cyclase (Ni) is present in 1321N1 cells. Pertussis toxin pretreatment of NG108-15 neuroblastoma X glioma cells, which express muscarinic receptors that link through Ni to inhibit adenylate cyclase, blocked the GTP-sensitive, high affinity binding of carbachol. In contrast, pretreatment of 1321N1 cells with a concentration of pertussis toxin that blocked [32P]ADP ribosylation of the Mr = 41,000 substrate and GTP-mediated inhibition of forskolin-stimulated adenylate cyclase activity had no effect on GTP-sensitive high affinity binding of carbachol. These results suggest that muscarinic cholinergic receptors of 1321N1 cells couple to a guanine nucleotide regulatory protein that is distinct from Ni.
Mol Pharmacol 1985 Jan
PMID:Guanine nucleotide-sensitive, high affinity binding of carbachol to muscarinic cholinergic receptors of 1321N1 astrocytoma cells is insensitive to pertussis toxin. 298

Considerably reduced responses to stimulation by isoproterenol of adenylate cyclase activity of prostatic membranes were observed in 12- to 18-month-old rats, compared to 3-month-old animals. Plasma testosterone levels were significantly lower in 18-month-old rats, while 12-month-old animals showed levels similar to those present in young ones. A decrease in isoproterenol activation of adenylate cyclase was not associated with a fall in beta-adrenergic receptor sites. Guanine triphosphate and 5'-guanylylimidodiphosphate (Gpp(NH)p) were effective in potentiation of isoproterenol activation of adenylate cyclase and altering the affinity of beta-adrenergic receptors for the agonist in membranes from young rats but not from the aged. The age-induced refractoriness to isoproterenol or Gpp(NH)p was observed without a significant loss in NaF-stimulated activity. Prior incubation of aged membranes with isoproterenol and GMP restored subsequent stimulation by Gpp(NH)p, presumably due to the clearance of inhibitory GDP tightly bound to the guanine nucleotide regulatory components in aged membranes. These results indicate that the dysfunction in the adenylate cyclase system of old prostates may not be related to a modification in the beta-adrenergic receptor per se, but to, in part, a defect in the interaction of activating guanine nucleotides with regulatory components of the adenylate cyclase system.
Mol Pharmacol 1985 Feb
PMID:Age-related alterations in the catecholamine-sensitive adenylate cyclase system of the prostate. 298 88


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