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

We have previously demonstrated that substitution of Asn for Ser at position 17 of RasH yields a dominant inhibitory protein whose expression in cells interferes with endogenous Ras function (L. A. Feig, and G. M. Cooper, Mol. Cell. Biol. 8:3235-3243, 1988). Subsequent structural studies have shown that the hydroxyl group of Ser-17 contributes to the binding of Mg2+ associated with bound nucleotide. In this report, we show that more subtle amino acid substitutions at this site that would be expected to interfere with complexing Mg2+, such as Cys or Ala, also generated dominant inhibitory mutants. In contrast, a Thr substitution that conserves a reactive hydroxyl group maintained normal Ras function. These results argue that the defect responsible for the inhibitory activity is improper coordination of Mg2+. Preferential affinity for GDP, observed in the original Asn-17 mutant, was found exclusively in inhibitory mutants. However, this binding specificity did not completely block the mutant proteins from binding GTP in vivo since introduction of the autophosphorylation site, Thr-59, in 17N Ras resulted in the phosphorylation of the double mutant in cells. Furthermore, inhibitory mutants failed to activate a model downstream target, yeast adenylate cyclase, even when bound to GTP. Thus, the consequence of improper complexing of Mg2+ was to lock the protein in a constitutively inactive state. A model is presented to explain how these properties could cause the mutant protein to inhibit the activation of endogenous Ras by competing for a guanine nucleotide-releasing factor.
Mol Cell Biol 1991 Oct
PMID:Dominant inhibitory mutations in the Mg(2+)-binding site of RasH prevent its activation by GTP. 192 22

The mechanism of the antiadrenergic action of adenosine in the heart was investigated by examining the effects of phenylisopropyladenosine (PIA), an adenosine A1 receptor agonist, on beta-adrenergic receptor and non-receptor elicited increases in adenylyl cyclase activity of guinea-pig ventricular membranes. These membranes contained adenosine A1 receptors (approximately 80 fmol/mg) and at least one ADP-ribosylated G protein with a molecular weight of approximately 40 kDa. PIA attenuated isoproterenol-enhanced adenylyl cyclase activity and [3H]GDP release in this membrane preparation. However, PIA had no significant effect on GPP(NP)P or forskolin activated adenylyl cyclase. Additionally, PIA did not change the sensitivity of the cyclase to either magnesium or GTP in these membranes. The inhibition of isoproterenol-enhanced activity appeared to be dependent on the activation state of the enzyme such that the degree of PIA inhibition decreased with increasing isoproterenol concentration. These data suggest that adenosine inhibition of catecholamine-stimulated adenylyl cyclase activity occurs predominantly by modulating beta-adrenergic receptor signal transduction and that subunits of Gi may be involved in this action.
J Mol Cell Cardiol 1990 Dec
PMID:Adenosine modulates beta-adrenergic signal transduction in guinea-pig heart ventricular membranes. 196 10

The product of the CDC25 gene of Saccharomyces cerevisiae, in its capacity as an activator of the RAS/cyclic AMP pathway, is required for initiation of the cell cycle. In this report, we provide an identification of Cdc25p, the product of the CDC25 gene, and evidence that it promotes exchange of guanine nucleotides bound to Ras in vitro. Extracts of strains containing high levels of Cdc25p catalyze both removal of GDP from and the concurrent binding of GTP to Ras. This same activity is also obtained with an immunopurified Cdc25p-beta-galactosidase fusion protein, suggesting that Cdc25p participates directly in the exchange reaction. This biochemical activity is consistent with previous genetic analysis of CDC25 function.
Mol Cell Biol 1991 May
PMID:The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras. 201 69

Previous work has shown that microinjection into cells of antibodies against p21ras blocks transformation by src, suggesting that oncogenic transformation by pp60v-src is dependent on p21ras. The activity of p21ras itself is regulated by its cyclic association with GDP-GTP, where p21ras-GTP is the active form and p21ras-GDP is the inactive form. A GTPase-activating protein (GAP) mediates the inactivation of p21ras by facilitating the conversion of the active p21ras-GTP to the inactive p21ras-GDP. This predicts that overexpression of GAP would inactivate p21ras and block transformation of cells by src. In this paper, we confirm this prediction. We report that overexpression of GAP in NIH 3T3 cells blocks transformation by pp60v-src but not by v-ras. Susceptibility to transformation by v-src is restored when GAP expression is lowered to levels comparable to that in control cells. These results support the suggestion that p21ras plays a central role in the signalling pathway used by pp60v-src.
Mol Cell Biol 1991 May
PMID:Inhibition of v-src-induced transformation by a GTPase-activating protein. 201 78

Loss of function of the Schizosaccharomyces pombe gap1 gene results in the same phenotypes as those caused by an activated ras1 mutation, i.e., hypersensitivity to the mating factor and inability to perform efficient mating. Sequence analysis of gap1 indicates that it encodes a homolog of the mammalian Ras GTPase-activating protein (GAP). The predicted gap1 gene product has 766 amino acids with relatively short N- and C-terminal regions flanking the conserved core sequence of GAP. Genetic analysis suggests that S. pombe Gap1 functions primarily as a negative regulator of Ras1, like S. cerevisiae GAP homologs encoded by IRA1 and IRA2, but is unlikely to be a downstream effector of the Ras protein, a role proposed for mammalian GAP. Thus, Gap1 and Ste6, a putative GDP-GTP-exchanging protein for Ras1 previously identified, appear to play antagonistic roles in the Ras-GTPase cycle in S. pombe. Furthermore, we suggest that this Ras-GTPase cycle involves the ra12 gene product, another positive regulator of Ras1 whose homologs have not been identified in other organisms, which could function either as a second GDP-GTP-exchanging protein or as a factor that negatively regulates Gap1 activity.
Mol Cell Biol 1991 Jun
PMID:Identification of a GTPase-activating protein homolog in Schizosaccharomyces pombe. 203 19

Purified porcine atrial muscarinic acetylcholine receptors were reconstituted into lipid vesicles with three different G proteins (Gi, Go and Gn)1 purified from porcine cerebrum. All the G proteins interacted with the receptor as evidenced by GTP-sensitive high affinity binding with acetylcholine, and stimulation by acetylcholine of GTP gamma S binding and GTPase activities. The curves of displacement by acetylcholine of [3H]QNB binding were explained by assuming two sites with the same affinity for [3H]QNB but different affinities for acetylcholine. The proportion of the high affinity site increased from 3 to 7% up to 82 to 83% of total binding sites with increasing G protein concentration, and essentially the same results were obtained with the three G proteins. The GTPase activities of Gi, Go and Gn in the reconstituted vesicles were 2.7-, 1.7- and 1.6-times higher, respectively, in the presence of 1 mM acetylcholine than those in the presence of 10 microM atropine. An obvious enhancement by acetylcholine of the GTP gamma S binding was observed in the presence of 10 to 100 microM GDP, while the enhancement was minimal, if at all, in the absence of GDP. When the molar ratios of reconstituted Gi, Go and Gn to muscarinic receptors were 54, 84 and 107, respectively, the acetylcholine-induced increase in the [35S]GTP gamma S binding was as much as 12, 35 and 27 mol with Gi, Go and Gn, respectively, per mole of the receptor molecule, indicating that the muscarinic receptors interact with G proteins catalytically.(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Cell Cardiol 1990 Mar
PMID:Interaction of atrial muscarinic receptors with three kinds of GTP-binding proteins. 211 1

Inhibition of bovine brain calmodulin-sensitive adenylyl cyclase was examined in a system consisting of the reconstituted purified porcine atrial muscarinic acetylcholine receptor, the purified inhibitory guanine nucleotide-binding protein (Gi), and the partially purified stimulatory guanine nucleotide-binding protein.adenylyl cyclase complex. Under conditions where Gi existed mainly as the Gi.GDP complex, adenylyl cyclase was selectively preactivated with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Addition of carbachol formed the receptor.carbachol complex, which catalyzed the exchange of GDP bound to Gi for GTP gamma S, initiating Gi-mediated inhibition of adenylyl cyclase. Adenylyl cyclase activated by calcium plus calmodulin was more sensitive to inhibition by carbachol than either unstimulated adenylyl cyclase or adenylyl cyclase activated by GTP gamma S or forskolin. Studies using the resolved subunits of Gi showed that the beta gamma subunit could inhibit adenylyl cyclase activated by GTP gamma S or calcium plus calmodulin, as well as the unactivated enzyme. The alpha subunit of Gi inhibited adenylyl cyclase only when adenylyl cyclase was activated by calcium plus calmodulin. Possible explanations for these results are discussed.
Mol Pharmacol 1990 Jun
PMID:Reconstitution of muscarinic receptor-mediated inhibition of adenylyl cyclase. 211 6

We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol. This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it. In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of purified smg p25A GDI. The cDNA has an open reading frame that encodes a protein of 447 amino acids with a calculated Mr of 50,565. This Mr is similar to those of the purified smg p25A GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation, which are about 54,000 and 65,000, respectively. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits GDI activity. smg p25A GDI is hydrophilic overall, except for one hydrophobic region near the N terminus. smg p25A GDI shares low amino acid sequence homology with the Saccharomyces cerevisiae CDC25-encoded protein, which has been suggested to serve as a factor that regulates the GDP-GTP exchange reaction of the yeast RAS2-encoded protein, but not with the beta gamma subunits of GTP-binding proteins having an alpha beta gamma subunit structure, such as Gs and Gi. The smg p25A GDI mRNA was present in various tissues, including not only tissues in which smg p25A was detectable but also tissues in which it was not detectable. This fact has raised the possibility that smg p25A GDI interacts with another G protein in tissues in which smg p25A is absent.
Mol Cell Biol 1990 Aug
PMID:Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein. 211 18

The calcium current components of acutely dissociated nodose ganglion neurons were characterized using the whole-cell variation of the patch-clamp technique. Many neurotransmitters regulate neuronal calcium currents via GTP binding (G) proteins and in some cases affect calcium current components selectively. To determine whether G proteins regulated these current components in the absence of ligand binding, recording pipettes contained 0.1 mM GTP, guanosine 5'-O-(thiodiphosphate) (GDP-beta-S), or guanosine 5'-O-(thiotriphosphate) (GTP-gamma-S). Nodose ganglion neurons had three calcium current components, similar to T, N, and L current components found in other sensory neurons. Isolated T currents did not diminish in magnitude during a 20-min recording, but there was a progressive loss of currents containing the N and L current components. The reduction of current magnitude was primarily dependent on the extent of intracellular dialysis and not on the holding potential (Vh) or stimulus frequency. When GDP-beta-S was substituted for GTP in the pipette solution, there was no change in the T current or in the rate of run-down of N and L current components. Substitution of GTP-gamma-S for GTP in the pipette solution resulted in a moderate (approximately 40%) loss of isolated T current. This effect was most evident on T currents evoked at relatively positive clamp potentials (Vc, -30 to -15 mV) and occurred relatively late (approximately 10 min) in the recording. In the presence of GTP-gamma-S, currents evoked from Vh = -80 mV, containing the N and L current components, were reduced 40-60%, with a lesser effect on those currents evoked from Vh = -40 mV, containing primarily the L current component. The average time to peak current (Ip) was increased 3-4-fold in the presence of GTP-gamma-S, and the Vc at which the maximal peak current was evoked was shifted +10 to 20 mV. These effects were evident within 2-5 min after initiation of the whole-cell recording. Pretreatment of neurons with pertussis toxin attenuated or blocked the effects of GTP-gamma-S. We conclude that nodose ganglion neurons have T-, N-, and L-type calcium current components, which had different stability during whole-cell recording. Activation of G proteins with GTP-gamma-S reduced N greater than T much greater than L currents, effects reduced in the presence of pertussis toxin. Thus, the calcium current components of nodose ganglion neurons were regulated by cytosolic constituents and by Gi- or Go- type G proteins.
Mol Pharmacol 1990 Apr
PMID:Regulation by GTP and its stable thiol derivatives of calcium current components in rat nodose ganglion neurons. 215 61

Normal human rap1A and 35A rap1A (which encodes a protein with a Thr-35----Ala mutation) were cloned into a baculovirus transfer vector and expressed in Sf9 insect cells. The resulting proteins were purified, and their nucleotide binding, GTPase activities, and responsiveness to GTPase-activating proteins (GAPs) were characterized and compared with those of Rap1 purified from human neutrophils. Recombinant wild-type Rap1A bound GTP gamma S, GTP, and GDP with affinities similar to those observed for neutrophil Rap1 protein. The rate of exchange of GTP by Rap1 without Mg2+ was much slower than that by Ras. The basal GTPase activities by both recombinant proteins were lower than that observed with the neutrophil Rap1, but the GTPase activity of the neutrophil and wild-type recombinant Rap1 proteins could be stimulated to similar levels by Rap-GAP activity in neutrophil cytosol. In contrast to wild-type Rap1A, the GTPase activity of 35A Rap was unresponsive to Rap-GAP stimulation. Neither recombinant Rap1A nor neutrophil Rap1 protein GTPase activity could be stimulated by recombinant Ras-GAP at a concentration 25-fold higher than that required to hydrolyze 50% of H-Ras-bound GTP under similar conditions. These results suggest that the putative effector domains (amino acids 32 to 40) shared between Rap1 and Ras are functionally similar and interact with their respective GAPs. However, although Rap1 and Ras are identical in this region, secondary structure or additional regions must confer the ability to respond to GAPs.
Mol Cell Biol 1990 Jun
PMID:Biochemical characterization of baculovirus-expressed rap1A/Krev-1 and its regulation by GTPase-activating proteins. 216 May 89


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