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Query: UNIPROT:P06889 (Mol)
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The antibodies described in the accompanying paper were used to probe the interactions of the chick heart muscarinic acetylcholine receptors (mAChRs) with the GTP-binding protein G(o). The anti-m4b antibodies, which were made against a peptide from the amino-terminal portion of the third cytoplasmic loop of the m4 mAChR subtype, were tested for their abilities to affect the coupling of the chick heart mAChR to the GTP-binding protein G(o). The purified chick heart mAChRs were reconstituted with purified G(o) in phospholipid vesicles, and their interactions were monitored in the presence or absence of the antibodies. The anti-m4b antibodies completely inhibited the ability of G(o) to promote high affinity agonist binding to the purified receptors. The anti-m4b antibodies also completely inhibited the agonist-stimulated binding of guanosine-5'-O-(3-thio)triphosphate (GTP gamma S) to G(o) and the receptor-stimulated GTPase activity of G(o). These findings indicate that the amino-terminal portion of the third cytoplasmic loop is an important determinant for G(o) to promote high affinity agonist binding to the chick heart mAChR and also for the agonist-stimulated GTP gamma S binding and GTPase activity. The anti-m4a, anti-m2, and anti-m1a antibodies, which were made against centrally located peptides of the third cytoplasmic loop of the m4, m2, and m1 mAChR subtypes, respectively, were also tested for their effects in the reconstituted receptor/G(o) system. The anti-m2 and anti-m4a antibodies also significantly reduced agonist-stimulated GTP gamma S binding, as well as GTPase activity, but did not completely abolish these functions, as was the case with anti-m4b antibodies. However, the anti-m4a and anti-m2 antibodies shared with anti-m4b antibodies the ability to markedly inhibit the ability of G(o) to promote high affinity agonist binding to the purified and reconstituted receptors. In contrast to the results obtained with the anti-m2 and anti-m4 antibodies, the anti-m1a antibodies had smaller effects on the receptor/G(o) interactions. These results suggested that central portions of the loop can also influence mAChR/G(o) interactions. Studies were als performed to test the effects of the peptides that were used as antigens on receptor-mediated GTP gamma S binding to G(o). Each of the peptides caused significant inhibition of this function, but the greatest inhibition was observed with the m4b peptide. In sum, the results suggest that multiple domains in the third cytoplasmic loop of chick heart mAChR can modulate interactions with G(o).
Mol Pharmacol 1991 Dec
PMID:Subtype-specific antibodies for muscarinic cholinergic receptors. II. Studies with reconstituted chick heart receptors and the GTP-binding protein G(o). 175 42

Three G proteins from human brain membranes were purified to near homogeneity by conventional techniques including preparative electrophoresis. These G proteins were characterized by their ability to bind GTP, GDP and GTP analogs. Two of these proteins have molecular weights of 50,000 (G50) and 36,000 (G36), as determined on SDS-gels. G36 was ADP-ribosylated by pertussis toxin. Thus, G50 could represent a Gs alpha subunit, whereas G36 could be Gi alpha or Go alpha. G50 was phosphorylated by cAMP dependent protein kinase and protein kinase C. G36 was phosphorylated by a protein kinase independent of calcium and phospholipid, a proteolytic product of protein kinase C, analogous to protein kinase M. Phosphorylation of G36 by this protein kinase induced a dramatic decrease in its GTPase activity. The third G protein, of molecular weight 22,000 probably belongs to the group of monomeric G proteins possessing functional similarities with ras gene products. The regulation of G proteins involving calcium-dependent and independent pathways is delineated.
Mol Cell Biochem 1991 Sep 18
PMID:Purification and characterization of G proteins from human brain: modification of GTPase activity upon phosphorylation. 178 75

The biological functions of ras proteins are controlled by the bound guanine nucleotide GDP or GTP. The GTP-bound conformation is biologically active, and is rapidly deactivated to the GDP-bound conformation through interaction with GAP (GTPase Activating Protein). Most transforming mutants of ras proteins have drastically reduced GTP hydrolysis rates even in the presence of GAP. The crystal structures of the GDP complexes of ras proteins at 2.2 A resolution reveal the detailed interaction between the ras proteins and the GDP molecule. All the currently known transforming mutation positions are clustered around the bound guanine nucleotide molecule. The presumed "effector" region and the GAP recognition region are both highly exposed. No significant structural differences were found between the GDP complexes of normal ras protein and the oncogenic mutant with valine at position 12, except the side-chain of the valine residue. However, comparison with GTP-analog complexes of ras proteins suggests that the valine side-chain may inhibit GTP hydrolysis in two possible ways: (1) interacting directly with the gamma-phosphate and altering its orientation or the conformation of protein residues around the phosphates; and/or (2) preventing either the departure of gamma-phosphate on GTP hydrolysis or the entrance of a nucleophilic group to attack the gamma-phosphate. The structural similarity between ras protein and the bacterial elongation factor Tu suggests that their common structural motif might be conserved for other guanine nucleotide binding proteins.
J Mol Biol 1991 Feb 05
PMID:Crystal structures at 2.2 A resolution of the catalytic domains of normal ras protein and an oncogenic mutant complexed with GDP. 189 7

smg p25A is a ras p21-like small GTP-binding protein which is implicated in the regulated secretory processes. We have recently found that bovine brain smg p25A is geranylgeranylated at its C-terminal region. In this study, we examined the function(s) of the C-terminal region of smg p25A. Limited proteolysis of bovine brain smg p25A with Achromobacter protease I produced an N-terminal fragment and a C-terminal tail. The Mrs of intact smg p25A, the N-terminal fragment, and the C-terminal tail were estimated to be about 24,000, 20,000, and less than 2,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal fragment contained the consensus amino acid sequences for GDP/GTP-binding and GTPase activities and showed these activities with kinetic properties similar to those of the intact protein but did not bind to plasma membranes or phosphatidylserine-linked Affigel under conditions in which the intact protein bound to them. The C-terminal tail neither contained the consensus amino acid sequences for GDP/GTP-binding and GTPase activities nor bound to plasma membranes or phosphatidylserine-linked Affigel. The GDP/GTP exchange protein specific for smg p25A, named GDP dissociation inhibitor (GDI), made a complex with the GDP-bound form of the intact smg p25A at a molar ratio of 1:1 and thereby inhibited its GDP/GTP exchange reaction but neither made a complex with the N-terminal fragment or the C-terminal tail nor affected the GDP/GTP exchange reaction of the N-terminal fragment. We expressed smg p25A in Escherichia coli and purified it to near homogeneity. This bacterial protein was not geranylgeranylated. Bacterial smg p25A did not bind to plasma membranes or phosphatidylserine-linked Affigel. smg p25A GDI neither made a complex with bacterial smg p25A nor affected its GDP/GTP exchange reaction. These results suggest that the N-terminal region of smg p25A has GDP/GTP-binding and GTPase activities but lacks the ability to interact with membranes and smg p25A GDI, that the C-terminal region of smg p25A plays important roles in its interaction with membranes and smg p25A GDI, and that some modifications of the C-terminal region, such as geranylgeranylation, which are absent in bacterial smg p25A, are important for these interactions.
Mol Cell Biol 1991 Mar
PMID:Role of the C-terminal region of smg p25A in its interaction with membranes and the GDP/GTP exchange protein. 189 8

The antibiotic thiostrepton, a thiazole-containing peptide, inhibits translation and ribosomal GTPase activity by binding directly to a limited and highly conserved region of the large subunit ribosomal RNA termed the GTPase center. We have previously used a filter binding assay to examine the binding of ribosomal protein L11 to a set of ribosomal RNA fragments encompassing the Escherichia coli GTPase center sequence. We show here that thiostrepton binding to the same RNA fragments can also be detected in a filter binding assay. Binding is relatively independent of monovalent salt concentration and temperature but requires a minimum Mg2+ concentration of about 0.5 mM. To help determine the RNA features recognized by L11 and thiostrepton, a set of over 40 RNA sequence variants was prepared which, taken together, change every nucleotide within the 1051 to 1108 recognition domain while preserving the known secondary structure of the RNA. Binding constants for L11 and thiostrepton interaction with these RNAs were measured. Only a small number of sequence variants had more than fivefold effects on L11 binding affinities, and most of these were clustered around a junction of helical segments. These same mutants had similar effects on thiostrepton binding, but more than half of the other sequence changes substantially reduced thiostrepton binding. On the basis of these data and chemical modification studies of this RNA domain in the literature, we propose that L11 makes few, if any, contacts with RNA bases, but recognizes the three-dimensional conformation of the RNA backbone. We also argue from the data that thiostrepton is probably sensitive to small changes in RNA conformation. The results are discussed in terms of a model in which conformational flexibility of the GTPase center RNA is functionally important during the ribosome elongation cycle.
J Mol Biol 1991 Oct 20
PMID:Recognition of the highly conserved GTPase center of 23 S ribosomal RNA by ribosomal protein L11 and the antibiotic thiostrepton. 194 50

The detergent-soluble extract of rat ovary plasma membranes contained a Gs protein of about 100 kDa as shown by its elution behavior on a Bio Gel A-1.5m column. However, the cell membranes exposed to hCG (37 degrees C, 15 min) contained in addition a higher molecular weight Gs protein complex of 300 kDa comprised of human chorionic gonadotropin (hCG) receptor (hCGR) and Gs. The complex bound with an affinity column of GTP-Sepharose and could be released with Gpp(NH)p and GTP inhibited this binding. The presence of the hCGR in the complex was shown by its binding to 125I-hCG. Furthermore, GTP inhibited the binding of hCG to the complex. These results indicate the presence of hCGR and Gs protein complex in the hCG-treated membranes. hCGR and Gs protein were individually purified and reconstituted into phospholipid vesicles. The protein-phospholipid vesicles showed saturation kinetics of binding of 125I-hCG and 3H-Gpp(NH)p. Incubation of phospholipid vesicles with hCG resulted in a 2-3-fold increase in the binding of 3H-Gpp(NH)p and GTPase activity. Activation of Gs protein was dependent on the length of incubation and the hormone concentration. Deglycosylated hCG was about 10 times less potent than hCG suggesting a role of carbohydrates of hCG in inducing hCG-Gs protein interactions. The data with the in vitro reconstitution system rule out the involvement of a carbohydrate-binding lectin in the function of the hormone.
Mol Cell Endocrinol 1991 Sep
PMID:Human choriogonadotropin-induced coupling of receptor and Gs protein and the effect of hormone deglycosylation. 195 70

The p21ras GTPase-activating protein (GAP) down-regulates p21ras by stimulating its intrinsic GTPase activity. GAP is found predominantly as a monomer in the cytosol of normal cells. However, in cells expressing an activated cytoplasmic protein-tyrosine kinase, p60v-src, or stimulated with epidermal growth factor, GAP becomes phosphorylated on tyrosine and serine and forms distinct complexes with two phosphoproteins of 62 and 190 kDa (p62 and p190). In v-src-transformed Rat-2 cells, a minor fraction of GAP associates with the highly tyrosine phosphorylated p62 to form a complex that is localized at the plasma membrane and in the cytosol. In contrast, the majority of GAP enters a distinct complex with p190 that is exclusively cytosolic and contains predominantly phosphoserine. Epidermal growth factor stimulation also induces a marked conversion of monomeric GAP to higher-molecular-weight species in rat fibroblasts. The GAP-p190 complex is dependent on phosphorylation and shows reduced GAP activity. These results indicate that protein-tyrosine kinases induce GAP to form multiple heteromeric complexes, which are strong candidates for regulators or targets of p21ras.
Mol Cell Biol 1991 Apr
PMID:Protein-tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution, and activity of p21ras GTPase-activating protein. 200 83

Overexpression of the full-length GTPase-activating protein (GAP) has recently been shown to suppress c-ras transformation of NIH 3T3 cells but not v-ras transformation (36). Here, we show that focus formation induced by c-src was inhibited by approximately 80% when cotransfected with a plasmid encoding full-length GAP. In a similar assay, focus formation by the activated c-src (Tyr-527 to Phe) gene was inhibited by 33%. Cotransfection of the GAP C terminus coding sequences (which encode the GTPase-accelerating domain) with c-src or c-src527F inhibited transformation more efficiently than did the full-length GAP, while the GAP N terminus coding sequences had no effect on src transformation. When cells transformed by c-ras, c-src, c-src527F, or v-src were transfected with GAP or the GAP C terminus sequence in the presence of a selectable marker, 40 to 85% of the resistant colonies were found to be morphologically revertant. The GAP C terminus induced reversion of each src-transformed cell line more efficiently than the full-length GAP, but this was not the case for reversion of c-ras transformation. Biochemical analysis of v-src revertant subclones showed that the reversion correlated with overexpression of full-length GAP or the GAP C terminus. There was no decrease in the level of pp60src expression or the level of protein-tyrosine phosphorylation in vivo. We conclude that GAP can suppress transformation by src via inhibition of endogenous ras activity, without inhibiting in vivo tyrosine phosphorylation of cellular proteins induced by pp60src, and that src may negatively regulate GAP's inhibitory action on endogenous ras.
Mol Cell Biol 1991 May
PMID:Suppression of src transformation by overexpression of full-length GTPase-activating protein (GAP) or of the GAP C terminus. 201 79

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

A series of v-rasH effector domain mutants were analyzed for their ability to transform rat 2 cells at either low or high temperatures. Three mutants were found to be significantly temperature sensitive: Ile-36 changed to Leu, Ser-39 changed to Cys (S39C), and Arg-41 changed to Leu. Of these, the codon 39 mutant (S39C) showed the greatest degree of temperature sensitivity. When the same mutation was analyzed in the proto-oncogene form of ras(c-rasH), this gene was also found to be temperature sensitive for transformation. Biochemical analysis of the proteins encoded by v-rasH(S39C) and c-rasH(S39C) demonstrated that the encoded p21ras proteins were stable and bound guanine nucleotides in vivo at permissive and nonpermissive temperatures. On the basis of these findings, it is likely that the temperature-sensitive phenotype results from an inability of the mutant (S39C) p21ras to interact properly with the ras target effector molecule(s) at the nonpermissive temperature. We therefore analyzed the interaction between the c-rasH(S39C) protein and the potential target molecules GTPase-activating protein (GAP) and the GAP-related domain of NF-1, on the basis of stimulation of the mutant p21ras GTPase activity by these molecules in vitro. Assays conducted across a range of temperatures revealed no temperature sensitivity for stimulation of the mutant protein, compared with that of authentic c-rasH protein. We conclude that for this mutant, there is a dissociation between the stimulation of p21ras GTPase activity by GAP and the GAP-related domain NF-1 and their potential target function. Our results are also consistent with the existence of a distinct, as-yet-unidentified effector for mammalian ras proteins.
Mol Cell Biol 1991 Jun
PMID:A ras effector domain mutant which is temperature sensitive for cellular transformation: interactions with GTPase-activating protein and NF-1. 203 22


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