A synthetic peptide corresponding to a sequence in the GTPase activating protein inhibits p21ras stimulation and promotes guanine nucleotide exchange

Amino acid sequence homology between the GTPase Activating Protein (GAP) and the GTP-binding regulatory protein, Gs alpha, suggests that a specific region of GAP primary structure (residues 891-898) may be involved in its stimulation of p21ras GTP hydrolytic activity (McCormick, F. [1989] Nature 340, 678-679). A peptide, designated p891, corresponding to GAP residues 891-906 (M891RTRVVSGFVFLRLIC906) was synthesized and tested for its ability to inhibit GAP-stimulated p21ras GTPase activity. At a concentration of 25 microM, p891 inhibited GAP activity approximately 50%. Unexpectedly, p891 also stimulated GTP binding to p21N-ras independent of GAP. This stimulation correlated with an enhancement of p21N-ras.GDP dissociation; an approximate 15-fold increase in the presence of 10 microM p891. In contrast, dissociation of the p21N-ras.GTP gamma S complex was unaffected by 10 microM p891. The p21N-ras.GDP complex was unresponsive to 100 microM mastoparan, a peptide toxin shown previously to accelerate GDP dissociation from the guanine nucleotide regulatory proteins, Gi and Go. p21H-ras, as well as the two p21H-ras effector mutants, Ala-38, and Ala-35, Leu-36, also exhibited increased rates of GDP dissociation in the presence of p891. Also tested were three ras-related GTP-binding proteins; rap, G25K and rac. The rap.-GDP complex was unaffected by 10 microM p891. Dissociation of the G25K- and rac.GDP complexes were enhanced slightly; approximately 1.3- and 1.8-fold over control, respectively. Thus, the inhibitory effect of p891 on GAP stimulation of p21ras suggests that amino acids within the region 891-906 of GAP may be essential for interaction with p21ras. In addition, p891 independently affects the nucleotide exchange properties of p21ras.     

Effects of Mg2+ and the beta gamma-subunit complex on the interactions of guanine nucleotides with G proteins

Mg2+ interacts with the alpha subunits of guanine nucleotide-binding regulatory proteins (G proteins) in the presence of guanosine-5'-[gamma-thio]triphosphate (GTP-gamma S) to form a highly fluorescent complex from which nucleotide dissociates very slowly. The apparent Kd for interaction of G alpha X GTP gamma S with Mg2+ is approximately 5 nM, similar to the Km for G protein GTPase activity X G beta gamma increases the rate of dissociation of GTP gamma S from G alpha X GTP gamma S or G alpha X GTP gamma S X Mg2+ at low concentrations of Mg2+. When the concentration of Mg2+ exceeds 1 mM, G beta gamma dissociates from G beta gamma X G alpha X GTP gamma S X Mg2+. Compared with the dramatic effect of Mg2+ on binding of GTP gamma S to G alpha, the metal has relatively little effect on the binding of GDP. However, G beta gamma increases the affinity of G alpha for GDP by more than 100-fold. High concentrations of Mg2+ promote the dissociation of GDP from G beta gamma X G alpha X GDP, apparently without causing subunit dissociation. The steady-state rate of GTP hydrolysis is strictly correlated with the rate of dissociation of GDP from G alpha under all conditions examined. Thus, there are at least two sites for interaction of Mg2+ with G protein-nucleotide complexes. Furthermore, binding of G beta gamma and GTP gamma S to G alpha is negatively cooperative, while the binding interaction between G beta gamma and GDP is strongly positive.     

Purification and characterization from bovine brain cytosol of a novel regulatory protein inhibiting the dissociation of GDP from and the subsequent binding of GTP to rhoB p20, a ras p21-like GTP-binding protein

A novel regulatory protein for the rho proteins (rhoA p21 and rhoB p20), belonging to a ras p21/ras p21-like small molecular weight (Mr) GTP-binding protein (G protein) superfamily, was purified to near homogeneity from bovine brain cytosol and characterized. This regulatory protein, designated here as GDP dissociation inhibitor (GDI) for the rho proteins (rho GDI), inhibited the dissociation of GDP from rhoB p20 and the binding of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to the GDP-bound form of rhoB p20 but not of that to the guanine nucleotide-free form. The Mr value of rho GDI was estimated to be about 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and from the S value, indicating that rho GDI is composed of a single polypeptide without a subunit structure. The isoelectric point was about pH 5.7. rho GDI made a complex with the GDP-bound form of rhoB p20 with a molar ratio of 1:1 but not with the GTP gamma S-bound or guanine nucleotide-free form. rho GDI did not stimulate the GTPase activity of rhoB p20 and by itself showed neither GTP gamma S-binding nor GTPase activity. rho GDI was equally active for rhoA p21 and rhoB p20 but was inactive for other ras p21/ras p21-like G proteins including c-Ha-ras p21, smg p25A, and smg p21. rho GDI activity was detected in the cytosol fraction of various rat tissues. These results indicate that, in mammalian tissues, there is a novel type of regulatory protein specific for the rho proteins that interacts with the GDP-bound form of the rho proteins and thereby regulates the GDP/GTP exchange reaction of the rho proteins by inhibiting the dissociation of GDP from and the subsequent binding of GTP to them. Since there is a GTPase-activating protein for the rho proteins stimulating the GTPase activity of the rho proteins in mammalian tissues, the rho proteins appear to be regulated at least by GTPase-activating protein and GDI in a dual manner.     

Guanine nucleotide binding properties of the mammalian RalA protein produced in Escherichia coli

The simian ralA cDNA was inserted in a ptac expression vector, and high amounts of soluble ral protein were expressed in Escherichia coli. The purified p24ral contains 1 mol of bound nucleotide/mol of protein that can be exchanged against external nucleotide. The ral protein exchanges GDP with a t 1/2 of 90 min at 37 degrees C in the presence of Mg2+, and has a low GTPase activity (0.07 min-1 at 37 degrees C). We have also studied its affinity for various guanine nucleotides and analogs. NMR measurements show that the three-dimensional environment around the nucleotide is similar in p21ras and p24ral. In addition to these studies on the wild-type ral protein, we used in vitro mutagenesis to introduce substitutions corresponding to the Val12, Val12 + Thr59, and Leu61 substitutions of p21ras. These mutant ral proteins display altered nucleotide exchange kinetics and GTPase activities, however, the effects of the substitutions are less pronounced than in the ras proteins. p24ralVal12 + Thr59 autophosphorylates on the substituted Thr, as a side reaction of the GTP hydrolysis, but the rate is much lower than those of the Thr59 mutants of p21ras. These results show that ras and ral proteins have similar structures and biochemical properties. Significant differences are found, however, in the contribution of the Mg2+ ion to GDP binding, in the rate of the GTPase reaction and in the sensitivity of these two proteins to substitutions around the phosphate-binding site, suggesting that the various "small G-proteins" of the ras family perform different functions.     

Partial purification and characterization of GDP dissociation stimulator (GDS) for the rho proteins from bovine brain cytosol

A novel type of regulatory proteins for the rho proteins (rhoA p21 and rhoB p20), ras p21-like small GTP-binding proteins (G proteins), are partially purified from bovine brain cytosol. These regulatory proteins, named rho GDP dissociation stimulator (GDS) 1 and -2, stimulate the dissociation of GDP from rhoA p21 and rhoB p20. rho GDS1 and -2 are inactive for other ras p21/ras p21-like small G proteins including c-Ha-ras p21, smg p21B, and smg p25A. Since we have previously shown that the rate limiting step for the GDP/GTP exchange reaction of the rho proteins is the dissociation of GDP from these proteins, the present results suggest that rho GDS1 and -2 stimulate the GDP/GTP exchange reaction of the rho proteins. rho GDS1 and -2 are distinct from the GAP- and GDI-types of regulatory proteins for the rho proteins previously purified from bovine brain cytosol. rho GAP stimulates the GTPase activity of the rho proteins and rho GDI inhibits the GDP/GTP exchange reaction of the rho proteins. The present results together with these earlier observations indicate that the rho proteins are regulated by at least three different types of regulatory proteins, GDS, GDI, and GAP.     

Stoichiometric interaction of smg p21 with its GDP/GTP exchange protein and its novel action to regulate the translocation of smg p21 between membrane and cytoplasm

We have previously purified a GDP/GTP exchange protein for smg p21A and -B, members of a ras p21/ras p21-like small GTP-binding protein superfamily. This regulatory protein, named smg p21 GDP dissociation stimulator (GDS), stimulates the dissociation of both GDP and GTP from and the subsequent binding of both GDP and GTP to smg p21s. We show here that smg p21 GDS forms a complex with both the GDP- and GTP-bound forms of smg p21B at a molar ratio of about 1:1. Both the GDP- and GTP-bound forms of smg p21B bound to membranes. smg p21 GDS inhibited this binding and moreover induced the dissociation of the prebound smg p21B from the membranes. These results indicate that smg p21 GDS stoichiometrically interacts with smg p21B and thereby regulates its GDP/GTP exchange reaction and its translocation between membranes and cytoplasm.     

Molecular cloning of the cDNA for stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like small GTP-binding proteins) and characterization of stimulatory GDP/GTP exchange protein.

We have recently purified to near homogeneity the stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like GTP-binding proteins) from bovine brain cytosol. This regulatory protein, named GDP dissociation stimulator (GDS), stimulates the GDP/GTP exchange reaction of smg p21s by stimulating the dissociation of GDP from and the subsequent binding of GTP to them. In this study, we have isolated and sequenced the cDNA of smg p21 GDS from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of the purified smg p21 GDS. The cDNA has an open reading frame encoding a protein of 558 amino acids with a calculated Mr value of 61,066, similar to the Mr of 53,000 estimated for the purified smg p21 GDS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits smg p21 GDS activity. smg p21 GDS is overall hydrophilic, but there are several short hydrophobic regions. The smg p21 GDS mRNA is present in bovine brain and various rat tissues. smg p21 GDS has low amino acid sequence homology with the yeast CDC25 and SCD25 proteins, which may regulate the GDP/GTP exchange reaction of the yeast RAS2 protein, but not with ras p21 GTPase-activating protein, the inhibitory GDP/GTP exchange proteins (GDP dissociation inhibitor) for smg p25A and rho p21s, and the beta gamma subunits of heterotrimeric GTP-binding proteins such as Gs and Gi.     

Functional interactions of stimulatory and inhibitory GDP/GTP exchange proteins and their common substrate small GTP-binding protein.

smg GDS and rho GDI are stimulatory and inhibitory GDP/GTP exchange proteins, respectively, for a group of ras p21-related small GTP-binding proteins (G proteins). rho p21 is a common substrate small G protein for both GDP/GTP exchange proteins. We examined here the functional interactions of these GDP/GTP exchange proteins with rho p21 as a substrate. smg GDS and rho GDI interacted with the GDP-bound form of rho p21 and thereby stimulated and inhibited, respectively, the dissociation of GDP. The inhibitory effect of rho GDI was much stronger than the stimulatory effect of smg GDS. The GDP-bound form of rho p21 formed a complex with rho GDI but not with smg GDS in their simultaneous presence. Since the content of smg GDS was generally less than that of rho GDI in cells, these results suggest that there is some mechanism to release the inhibitory action of rho GDI and to make rho p21 sensitive to the smg GDS action during the conversion of rhoA p21 from the GDP-bound inactive form to the GTP-bound active form in intact cells. On the other hand, rho p21 was previously shown to be ADP-ribosylated by bacterial ADP-ribosyltransferases, named C3 and EDIN, at Asn41 in the putative effector region of rho p21. This ADP-ribosylation was inhibited by rho GDI much more efficiently than by smg GDS. These results suggest that rho GDI may mask the putative effector region of rho p21 and thereby inhibit its interaction with the target protein even in the presence of smg GDS. Thus, both smg GDS and rho GDI are important to regulate the rho p21 activity and action in cooperation with each other.     

Characterization of nucleoside-diphosphate kinase-associated guanine nucleotide-binding proteins from HeLa S3 cells

Nucleoside-diphosphate (NDP) kinase-associated [alpha-32P]GTP-incorporating proteins from HeLa S3 cells have been biochemically characterized. Two distinct NDP-kinases (F-I and F-II) had been partially purified from HeLa S3 cells by Sephacryl S-300 gel filtration and DEAE-cellulose column chromatography. The [alpha-32P]GTP-incorporating proteins (approx. Mr 20,000) could be separated from NDP-kinases (approx. Mr 80,000) by 5-25% glycerol density-gradient centrifugation analysis after treatment with 7 M urea in the presence of 1 mM EDTA. [alpha-32P]GTP incorporation into these two proteins (G1 and G2) from NDP-kinases required 5 mM Mg2+ and was highly inhibited by either GDP or GTP analogues, such as guanylyl imidodiphosphate and guanylyl methylenediphosphate. [3H]GDP, but no other nucleoside 5'-diphosphates, was also bound to these two proteins in the presence of Mg2+ (5 mM). Moreover, incubation of [alpha-32P]GTP with either G1 or G2 in the presence of Mg2+ (5 mM) resulted in the formation of [32P]GDP and Pi. The data presented here indicated that the guanine nucleotide-binding activity, the GTPase activity, and the molecular weight (approx. Mr 20,000) of NDP-kinase-associated proteins from HeLa S3 cells are similar to those reported for ras oncogene products (p21 proteins).     

Regulation of p21ras activity.

The ras genes encode GTP/GDP-binding proteins that participate in mediating mitogenic signals from membrane tyrosine kinases to downstream targets. The activity of p21ras is determined by the concentration of GTP-p21ras, which is tightly regulated by a complex array of positive and negative control mechanisms. GAP and NF1 can negatively regulate p21ras activity by stimulating hydrolysis of GTP bound to p21ras. Other cellular factors can positively regulate p21ras by stimulating GDP/GTP exchange.     

Purification and characterization from rat liver cytosol of a GDP dissociation inhibitor (GDI) for liver 24K G, a ras p21-like GTP-binding protein, with properties similar to those of smg p25A GDI

A regulatory protein for a liver GTP-binding protein (G protein) with a molecular weight value of 24,000 (24K G), which we have recently purified, was purified to near-homogeneity from rat liver cytosol and characterized. This regulatory protein, designated here as GDP dissociation inhibitor for 24K G (24K G GDI), inhibited the dissociation of GDP from and the subsequent binding of GTP to 24K G. 24K G GDI was inactive for other ras p21/ras p21-like small G proteins including c-Ha-ras p21, rhoB p20, smg p21B, and smg p25A. 24K G was, however, recognized by bovine brain smg p25A GDI which regulated the GDP/GTP exchange reaction of smg p25A. By analyses of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), immunoblotting with anti-smg p25A GDI antibody, two-dimensional PAGE, and C4 column chromatography, 24K G GDI showed physical properties very similar to those of smg p25A GDI. The peptide map and the partial amino acid sequences of 24K G GDI were not identical with those of smg p25A GDI. Among the 83 residues, 2 amino acids were different between rat liver 24K G GDI and bovine brain smg p25A GDI. These results indicate that there is a specific regulatory protein for 24K G, 24K G GDI, in rat liver cytosol and that 24K G GDI has close similarity to smg p25A GDI.     

Properties of the exchange rate of guanine nucleotides to the novel rap-2B protein

Rap-2B is a novel ras-related protein that is 89% identical to rap-2 at the amino acid level. Based on its amino acid sequence, it is anticipated that rap-2B binds guanine nucleotides. Here we show that purified, bacterially expressed rap-2B does bind both GTP and GDP in a Mg2(+)-dependent fashion. The relative affinity of rap-2B for GTP is higher than that for GDP, both at low and high concentrations of Mg2+. This contrasts with N-ras p21 and could be of functional significance. Moreover, a polyclonal antiserum was raised against the recombinant rap-2B protein purified from E. coli lysates. This antiserum recognized a major protein of Mr approximately 21000 on Western blots of platelet membrane proteins, and immunoprecipitates rap-2B complexed with GTP or GDP.     

The product of the rap2 gene, member of the ras superfamily. Biochemical characterization and site-directed mutagenesis

The human rap2 gene encodes a 183 amino acid protein that shares 46% identity with the K-ras p21. Its cDNA was engineered and inserted into the bacterial expression vector ptac; this allowed the production of high levels of soluble recombinant protein in Escherichia coli that was purified to near homogeneity. The rap2 protein binds GTP and exhibits a low intrinsic GTPase activity (rate constant of 0.5 x 10(-2) min-1). It exchanges its bound GDP with a half-life of 18 min at 37 degrees C in the presence of 10 mM Mg2+. Under the same conditions, the dissociation of bound GTP was at least 25-fold slower showing that the rap2 protein has a much higher affinity for GTP than GDP. The contribution of individual domains of the protein to its biochemical activities was investigated by site-directed mutagenesis. Substitution of Val for Gly at position 12 results in a 2-fold decrease in the GDP dissociation rate constant and GTPase activity. Replacement of the Ser at position 17 by Asn severely impairs the GTP binding ability of the protein and points to an important role of this residue in the coordination of Mg2+. Mutation of Thr-35 to Ala results in a decreased affinity for GTP and a reduction (3-fold) of the GTPase activity. Finally, substitution of Thr-145 by Ile leads to an imperfect binding of guanyl nucleotides as exemplified by an increase in their dissociation rate constants and reduction of the GTPase activity of the protein. These properties of the normal and mutant rap2 proteins are compared with those of ras p21 carrying similar substitutions and are discussed in relation to the structural models proposed for ras p21.     

Rapid irreversible G protein alpha subunit misfolding due to intramolecular kinetic bottleneck that precedes Mg2+ "lock" after GTP/GDP exchange

Stoichiometric exchange of GTP for GDP on heterotrimeric G protein alpha (Galpha) subunits is essential to most hormone and neurotransmitter initiated signal transduction. Galphas are stably activated in a Mg2+ complex with GTPgammaS, a nonhydrolyzable GTP analogue that is reported to bind Galpha, with very high affinity. Yet, it is common to find that substantial amounts (30-90%) of purified G proteins cannot be activated. Inactivatable G protein has heretofore been thought to have become "denatured" during formation of the obligatory nucleotide-free or empty (MT) Galpha-state that is intermediary to GDP/GTP exchange at a single binding site. We find Galpha native secondary and tertiary structure to persist during formation of the irreversibly inactivatable state of transducin. MT Galpha is therefore irreversibly misfolded rather than denatured. Inactivation by misfolding is found to compete kinetically with protective but weak preequilibrium nucleotide binding at micromolar ambient GTPgammaS concentrations. Because of the weak preequilibrium, quantitative protection against Galpha aggregation is only achieved at free nucleotide concentrations 10-100 times higher than those commonly employed in G protein radio-nucleotide binding studies. Initial GTP protection is also poor because of the extreme slowness of an intramolecular Galpha refolding step (isomerization) necessary for GTP sequestration after its weak preequilibrium binding. Of the two slowly interconverting Galpha x GTP isomers described here, only the second can bind Mg2+, "locking" GTP in place with a large net rise in GTP binding affinity. A companion Galpha x GDP isomerization reaction is identified as the cause of the very slow spontaneous GDP dissociation that characterizes G protein nucleotide exchange and low spontaneous background activity in the absence of GPCR activation. Galpha x GDP and Galpha x GTP isomerization reactions are proposed as the dual target for GPCR catalysis of nucleotide exchange.     

Kinetic analysis of the binding of guanine nucleotides to bovine brain rhoB p20, a ras p21-like GTP-binding protein

We have investigated the kinetics of the binding of guanine nucleotides to bovine brain rhoB p20, a ras p21-like GTP-binding protein with GTPase activity. The initial velocities of the binding of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to GDP-bound rhoB p20 and the dissociation of GDP from this protein were markedly increased by decreasing Mg2+ concentrations. The initial velocity of the binding of GTP gamma S to GDP-free rhoB p20 was not affected by changing Mg2+ concentrations. These results indicate that the dissociation of GDP from rhoB p20 limits the binding of GTP to this protein, and suggest that there is a factor stimulating the dissociation of GDP from rhoB p20 and thereby stimulating the binding of GTP to this protein in mammalian tissues. Consistently, the factor stimulating the dissociation of GDP, but not of GTP gamma S, from rhoB p20 was detected in bovine brain cytosol.     

Regeneration of the GTP-bound from the GDP-bound form of human and yeast ras proteins by nucleotide exchange. Stimulatory effect of organic and inorganic polyphosphates

The regeneration of the GTP-bound from the GDP-bound form of purified human and yeast ras proteins occurs in vitro by a nucleotide-exchange reaction. For both human and yeast ras proteins the dissociation of the protein-bound GDP is the rate-limiting step in the presence of Mg ions. The rate of formation of the ras X GTP complex is stimulated by weak Mg2+-chelating agents like ATP and inorganic polyphosphates and, to a lesser extent, by ADP. This suggests a possible mechanism of regulation of ras-dependent pathway(s) by intracellular metabolic products.     

Rabbit intestine contains a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to rhoB p20, a ras p21-like GTP-binding protein

A novel regulatory protein for rhoB p20, a ras p21-like GTP-binding protein (G protein), was partially purified from the cytosol fraction of rabbit intestine. This protein, designated as rhoB p20 GDP dissociation inhibitor (GDI), inhibited the dissociation of GDP from rhoB p20. rhoB p20 GDI also inhibited the binding of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to the GDP-bound form of rhoB p20 but not of that to the guanine nucleotide-free form. GDI did not affect the GTPase activity of rhoB p20 and by itself showed no GTP gamma S-binding activity. GDI was inactive for other ras p21/ras p21-like G proteins including c-Ha-ras p21, smg p21 and smg p25A. The Mr value of GDI was estimated to be about 27,000 from the S value. These results indicate that rabbit intestine contains a novel regulatory protein that inhibits the dissociation of GDP from and thereby the subsequent binding of GTP to rhoB p20.     

Cooperative function of rho GDS and rho GDI to regulate rho p21 activation in smooth muscle.

The GDP/GTP exchange reaction of rho p21, a member of ras p21-related small GTP-binding protein superfamily, is regulated by two stimulatory GDP/GTP exchange proteins (GEPs), named smg GDS and rho GDS, and by one inhibitory GEP, named rho GDI. In bovine aortic smooth muscle, rho GDS and rho GDI were major GEPs for rho p21, and the rho GDI activity on the GDP/GTP exchange reaction of rho p21 was stronger than the rho GDS activity in their simultaneous presence. Moreover, in the crude cytosol, the GDP-bound form of rho p21 was complexed with rho GDI but not with rho GDS. These results, together with our recent finding that rho p21 is involved in the vasoconstrictor-induced Ca2+ sensitization of smooth muscle contraction, suggest that there is some mechanism to release the inhibitory action of rho GDI and to make rho p21 sensitive to the stimulatory action of rho GDS, eventually leading to the rho p21 activation, in the signaling pathways of the vasoconstrictor receptors in smooth muscle.     

A small GTP-binding protein (G protein) recognized by smg p25A GDP dissociation inhibitor (GDI) in human platelet membranes and GDI for this small G protein in human platelet cytosol

We have recently purified from bovine brain cytosol a novel type of regulatory protein for smg p25A, named smg p25A GDP dissociation inhibitor (GDI), that regulates the GDP/GTP exchange reaction of smg p25A by inhibiting the dissociation of GDP from and thereby the subsequent binding of GTP to it. This smg p25A GDI is inactive for other ras p21/ras p21-like small GTP-binding proteins (G proteins) including c-Ha-ras p21, smg p21, rhoA p21 and rhoB p20. In human platelet membranes, smg p25A was not detected but a G protein with an apparent Mr value of 24,000 (24KG) was recognized by smg p25A GDI and the dissociation of GDP from and the binding of GTP to 24KG were inhibited by smg p25A GDI. The doses of smg p25A GDI necessary for these activities for both 24KG and smg p25A were the same. This 24KG was not recognized by an anti-smg p25A monoclonal antibody. The GDI activity for human platelet 24KG and smg p25A was detected in human platelet cytosol. This human platelet GDI was recognized by an anti-smg p25A GDI polyclonal antibody. These results indicate that there is a 24KG-24KG GDI system similar to a smg p25A-smg p25A GDI system in human platelets.     

The ral gene: a new ras related gene isolated by the use of a synthetic probe.

We synthesized a set of 20-mer oligonucleotides corresponding to a sequence of seven amino acids strictly conserved in all the different ras proteins, from yeast to man, as well as in rho and YPT, two proteins distantly related to p21 ras (approximately 30% amino acid homology). This oligonucleotide probe was used to search for new members of the ras family. We describe here a new ras related gene named ral, isolated from a cDNA library of immortalized simian B-lymphocytes. The ral gene codes for a 206 amino acid protein of expected mol. wt 23.5 kd that shares greater than 50% homology with H-ras, K-ras or N-ras. The GTP binding regions of p21 ras and a C-terminal cysteine involved in membrane anchoring are also present in ral; this strongly suggests that ral is a GTP binding protein with membrane localization. Furthermore, several external regions of p21 ras presumably involved in the interaction with effector, receptor and/or regulatory proteins are highly homologous to the corresponding regions in ral. Therefore some of the proteins that interact with ral might be identical or closely related to those interacting with p21 ras.