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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

cAMP receptor 1 and G-protein alpha-subunit 2 null cell lines (car1- and g alpha 2-) were examined to assess the roles that these two proteins play in cAMP stimulated adenylyl cyclase activation in Dictyostelium. In intact wild-type cells, cAMP stimulation elicited a rapid activation of adenylyl cyclase that peaked in 1-2 min and subsided within 5 min; in g alpha 2- cells, this activation did not occur; in car1- cells an activation occurred but it rose and subsided more slowly. cAMP also induced a persistent activation of adenylyl cyclase in growth stage cells that contain only low levels of cAMP receptor 1 (cAR1). In lysates of untreated wild-type, car1-, or g alpha 2- cells, guanosine 5'-O-'(3-thiotriphosphate) (GTP gamma S) produced a similar 20-fold increase in adenylyl cyclase activity. Brief treatment of intact cells with cAMP reduced this activity by 75% in control and g alpha 2- cells but by only 8% in the car1- cells. These observations suggest several conclusions regarding the cAMP signal transduction system. 1) cAR1 and another cAMP receptor are linked to activation of adenylyl cyclase in intact cells. Both excitation signals require G alpha 2. 2) cAR1 is required for normal adaptation of adenylyl cyclase. The adaptation reaction caused by cAR1 is not mediated via G alpha 2. 3) Neither cAR1 nor G alpha 2 is required for GTP gamma S-stimulation of adenylyl cyclase in cell lysates. The adenylyl cyclase is directly coupled to an as yet unidentified G-protein.
Mol Biol Cell 1992 Nov
PMID:Multiple cyclic AMP receptors are linked to adenylyl cyclase in Dictyostelium. 133 42

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

A new method based on the analysis of oligopeptide composition of the amino acid sequences from different protein families is presented. We assume, that any protein family can be characterized by the set of oligopeptides (oligopeptides vocabulary). We demonstrate, that oligopeptides vocabulary comparison can distinguish different families from each other and from random sequences. It should be noted, that this comparison can be successfully performed on the set of only 25 dipeptides and without preliminary alignment. We demonstrate, that characteristic peptides are localized in the regions of functional significance, as shown on the example of GTP-binding domain of translation elongation factors. We suggest how to use this method to localize the boundaries of functional domains in amino sequences. On the example of few functional domains we demonstrate, that the average error of prediction does not exceed 3-4 amino acid residue.
Mol Biol (Mosk)
PMID:[Search for functional domain boundaries and localization of functional sites based on oligopeptide vocabularies]. 133 54

In SK-N-SH human neuroblastoma cells, the muscarinic agonist carbachol promotes polyphosphoinositide (PPI) hydrolysis via M3 receptors and increases cyclic AMP levels through an unidentified mechanism. Activation of PPI hydrolysis by carbachol elicits a robust translocation of CaM from membranes into cytosol which was previously shown to be mimicked by the addition of the calcium ionophore ionomycin and the phorbol ester TPA28. The effect of agonist-stimulated second messenger production on CaM localization was determined by activating receptors that increase and decrease adenylyl cyclase activity on SK-N-SH cells. VIP (10 microM), prostaglandin E1 (30 microM) and forskolin (10 microM) all increased adenylyl cyclase activity 8- to 10-fold above the activity with 1 microM GTP. Carbachol (100 microM) did not stimulate adenylyl cyclase activity. The alpha 2-adrenergic agonist UK 14,304 (0.1 microM) and the delta and mu opioid DPDPE (10 microM) and DAMGO (10 microM) inhibited forskolin-stimulated cyclic AMP formation by 27-32%. CaM did not stimulate adenylyl cyclase activity. Incubation of cells with vasoactive intestinal polypeptide (VIP), dibutyryl cyclic AMP and forskolin, resulted in 30% decrease in membrane CaM and an increase in cytosolic CaM of 40-50%. The CaM translocation with the combination of an agent that elevates cyclic AMP levels and a low dose of carbachol was not different from that observed with either agent alone. UK 14,304, DPDPE and DAMGO potentiated carbachol-stimulated increases in cytosolic CaM. Upon the addition of carbachol, a 5-fold increase in intracellular calcium concentration measured with fura-2 fluorescence was observed. VIP and UK 14,304 elevated intracellular calcium concentrations 2 to 3 fold, while forskolin (10 microM) had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)
Brain Res Mol Brain Res 1992 Jan
PMID:Cyclic AMP accumulation alters calmodulin localization in SK-N-SH human neuroblastoma cells. 134 31

Conditions were established for the self-assembly of milligram amounts of purified Saccharomyces cerevisiae tubulin. Microtubules assembled with pure yeast tubulin were not stabilized by taxol; hybrid microtubules containing substoichiometric amounts of bovine tubulin were stabilized. Yeast microtubule-associated proteins (MAPs) were identified on affinity matrices made from hybrid and all-bovine microtubules. About 25 yeast MAPs were isolated. The amino-terminal sequences of several of these were determined: three were known metabolic enzymes, two were GTP-binding proteins (including the product of the SAR1 gene), and three were novel proteins not found in sequence databases. Affinity-purified antisera were generated against synthetic peptides corresponding to two of the apparently novel proteins (38 and 50 kDa). Immunofluorescence microscopy showed that both these proteins colocalize with intra- and extranuclear microtubules in vivo.
Mol Biol Cell 1992 Jan
PMID:Yeast proteins associated with microtubules in vitro and in vivo. 134 5

beta-Adrenergic receptor (beta AR) subtypes differ in their affinities for some agonists and antagonists and thus may potentially impart different cellular effects based on this ligand-binding specificity. However, the possibility that there may be subtype-specific events subsequent to ligand binding has not been evaluated extensively. In particular, although beta ARs stimulate adenylyl cyclase by coupling to the guanine nucleotide-binding protein Gs, no studies have directly assessed the coupling efficiencies among isolated beta AR subtypes. We, therefore, permanently transfected the mammalian fibroblast cell line CHW-1102 with beta 1- or beta 2AR cDNAs and studied the coupling characteristics of these two receptor subtypes, each expressed at approximately 335 fmol/mg of protein. Both receptors mediated equivalent maximal increases in adenylyl cyclase activities (6.63 +/- 1.85-fold for beta 1AR versus 6.10 +/- 0.53-fold for beta 2AR; p = not significant). However, the isoproterenol dose-response curves for the beta 2AR were shifted to the left, compared with those for the beta 1AR (EC50 of 52.3 +/- 2.87 nM and 191 +/- 10.5 nM, respectively; p less than 0.05), resulting in an approximately 4-fold greater potency for the beta 2AR versus the beta 1AR. Thus, at the submaximal isoproterenol concentration of 30 nM, the beta 2AR stimulated adenylyl cyclase approximately 50% more than did the beta 1AR. This finding was not due to a difference in the affinities of isoproterenol for these receptors, which were found to be the same, as determined by competition binding studies with 125I-cyanopindolol in the presence of GTP. The ability of beta 1- and beta 2ARs to form the high affinity ternary complex was assessed in agonist competition studies without guanine nucleotide. We found that, whereas the proportion of receptors in the high affinity state was equivalent between the two receptor subtypes, the affinity of this state for isoproterenol was approximately 5-fold greater for the beta 2AR, compared with the beta 1AR (KH for beta 2AR, 11.8 +/- 3.1 nM; KH for beta 1AR, 61.7 +/- 18.3 nM; p less than 0.05). In addition, we examined physical and functional coupling of beta 1- and beta 2ARs to Gs using the agonist epinephrine, which also has equal binding affinity for both receptor subtypes. As with isoproterenol, epinephrine was more potent in stimulating adenylyl cyclase and promoted a higher affinity ternary complex for the beta 2AR. Thus, a greater degree of both physical and functional agonist-promoted coupling occurs between Gs and beta 2AR, compared with beta 1AR. We conclude that coupling to Gs by beta 1- and beta 2ARs is subtype selective and is a potentially important distinguishing feature among these members of the beta AR family.
Mol Pharmacol 1992 May
PMID:Beta 1- and beta 2-adrenergic receptors display subtype-selective coupling to Gs. 135 Mar 21

In an attempt to study potential feedback regulation of the neu oncogene, we have found that the neu oncogene product specifically represses its own promoter activity. Deletion analysis indicated a 140-bp region (nucleotides -312 to -173 relative to the ATG initiation codon) in the rat neu promoter responsible for neu autorepression. Gel shift assays and methylation interference analysis further demonstrated that a GGTGGGGGGG sequence (nucleotides -243 to -234 relative to the ATG initiation codon) in this 140-bp region interacts with specific protein complexes. The GGTGGGGGGG sequence (GTG element), which functions as an enhancer, is sufficient to cause neu-mediated repression in a heterologous promoter. Furthermore, it produces different gel shift patterns with nuclear extracts from neu-transformed cell lines and their parental lines, suggesting that a transcriptional factor(s) interacting with this enhancer element has been perturbed by the introduction of neu. Taken together, the data presented in this report show that (i) the neu oncogene product autorepresses its own promoter, (ii) the neu promoter contains a novel enhancer, and (iii) neu autorepression is mediated through this enhancer, likely by inhibition of the enhancer activity.
Mol Cell Biol 1992 Jun
PMID:Negative autoregulation of the neu gene is mediated by a novel enhancer. 135 Mar 24

The structure of microtubules has been characterized to 3 nm resolution employing time-resolved X-ray scattering. This has revealed detailed structural features of microtubules not observed before in solution. The polymerization of highly purified tubulin, induced by the antitumour drug taxol, has been employed as a microtubule model system. This assembly reaction requires Mg2+, is optimal at a 1:1 taxol to tubulin heterodimer molar ratio, proceeds with GTP or GDP and is intrinsically reversible. The X-ray scattering profiles are consistent with identical non-globular alpha and beta-tubulin monomers ordered within the known helical surface lattice of microtubules. Purified tubulin-taxol microtubules have a smaller mean diameter (approx. 22 nm) than those induced by microtubule associated proteins or glycerol (approx. 24 nm), but nearly identical wall substructure to the resolution of the measurements. This is because the majority of the former consist of only 12 protofilaments instead of the typical 13 protofilaments, as confirmed by electron microscopy of thin-sectioned, negatively stained and ice-embedded taxol microtubules. It may be concluded that taxol induces a slight reduction of the lateral contact curvature between tubulin monomers. The main fringe pattern observed in cryo-electron micrographs is consistent with a simple 12 protofilament 3-start skewed lattice model. Cylindrical closure of this lattice can be achieved by tilting the lattice 0.8 degrees with respect to the microtubule axis. The closure implies a discontinuity in the type of lateral contacts between the tubulin monomers (regardless of whether these are of the -alpha-beta- or the -alpha-alpha-/-beta-beta- type), which indicates that lateral contacts and the subunit specificity of taxol binding are, to a large degree, equivalent.
J Mol Biol 1992 Jul 05
PMID:Low resolution structure of microtubules in solution. Synchrotron X-ray scattering and electron microscopy of taxol-induced microtubules assembled from purified tubulin in comparison with glycerol and MAP-induced microtubules. 135 57

The brain cyclic AMP generation was studied in rats subjected to 15 min of cardiac arrest. We have used a particulate, synaptoneurosomal fraction to demonstrate the effect of ischemia in vivo on the responsiveness of adenylate cyclase (AC) system. It has been shown that, although there is a slight decrease in AC activity after ischemia, the in vitro fractions produce more cAMP in response to a variety of stimuli, suggesting an indirect, nonadenylate cyclase activation mechanism. For elucidation of this mechanism we have probed phorbol-12,13-dibutyrate (PDBu) as a direct PKC activator, forskolin to activate the catalytic subunit of AC, and cholera toxin (CT) for stabilizing the active, GTP-bound form of stimulatory guanine nucleotide binding protein (Gs). All these postreceptor AC modulators as well as the receptor activators such as adenosine and alpha 1-adrenergic agonists markedly enhanced cAMP production in the rat brain particulate fraction, although the postischemic hyperactive response to these stimuli was still present. However, when AC was stimulated by the combination of CT and PDBu, cAMP responses were identical in both control and postischemic fractions. The data, taken together, support the hypothesis that ischemia increases cAMP accumulation by facilitating the postreceptor AC activation through a PKC-involving pathway and by promoting the stronger coupling of membrane AC receptors with G-protein. Protein kinase C (PKC) activity during cerebral ischemia was also investigated. In contradistinction to our expectation PKC decreased significantly in the ischemic brain to 85% of the control activity in the cytosol and 72% in the membranes. However, in the incubated post-ischemic brain particulate fraction a relative increase in the membrane-bound form of the enzyme, from 30% for control to 53% for ischemia, was observed. This may suggest that ischemia-induced membrane changes could promote the enzyme translocation/activation during recovery, resulting in the sensitization of cAMP producing system.
Mol Chem Neuropathol 1992 Aug
PMID:Postreceptor modulation of cAMP accumulation in rat brain particulate fraction after ischemia--involvement of protein kinase C. 135 40


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