Molecular cloning, bacterial expression and properties of Rab31 and Rab32.

GTP-binding proteins of the Rab family were cloned from human platelets using RT-PCR. Clones corresponding to two novel Rab proteins, Rab31 and Rab32, and to Rab11A, which had not been detected in platelets previously, were isolated. The coding sequence of Rab31 (GenBank accession no. U59877) corresponded to a 194 amino-acid protein of 21.6 kDa. The Rab32 sequence was extended to 1000 nucleotides including 630 nucleotides of coding sequence (GenBank accession no. U59878) but the 5' coding sequence was only completed later by others (GenBank accession no. U71127). Human Rab32 cDNA encodes a 225 amino-acid protein of 25.0 kDa with the unusual GTP-binding sequence DIAGQE in place of DTAGQE. Northern blots for Rab31 and Rab32 identified 4.4 kb and 1.35 kb mRNA species, respectively, in some human tissues and in human erythroleukemia (HEL) cells. Rabbit polyclonal anti-peptide antibodies to Rab31, Rab32 and Rab11A detected platelet proteins of 22 kDa, 28 kDa and 26 kDa, respectively. Human platelets were highly enriched in Rab11A (0.85 microg x mg of platelet protein(-1)) and contained substantial amounts of Rab32 (0.11 microg x mg protein(-1)). Little Rab31 was present (0.005 microg x mg protein(-1)). All three Rab proteins were found in both granule and membrane fractions from platelets. In rat platelets, the 28-kDa Rab32 was replaced by a 52-kDa immunoreactive protein. Rab31 and Rab32, expressed as glutathione S-transferase (GST)-fusion proteins, did not bind [alpha-(32)P]GTP on nitrocellulose blots but did bind [(35)S]GTP[S] in a Mg(2+)-dependent manner. Binding of [(35)S]GTP[S] was optimal with 5 microm Mg(2+)(free) and was markedly inhibited by higher Mg(2+) concentrations in the case of GST-Rab31 but not GST-Rab32. Both proteins displayed low steady-state GTPase activities, which were not inhibited by mutations (Rab31(Q64L) and Rab32(Q85L)) that abolish the GTPase activities of most low-M(r) GTP-binding proteins.     

Low molecular weight GTP-binding proteins in cardiac muscle. Association with a 32-kDa component related to connexins.

Low molecular weight GTP-binding proteins and their cellular interactions were examined in cardiac muscle. Heart homogenate was separated into various subcellular fractions by differential and sucrose density gradient centrifugation. Various fractions were separated by sodium dodecyl sulfate-gel electrophoresis, blotted to nitrocellulose, and GTP-binding proteins detected by incubating with [alpha-32]GTP. Three polypeptides of M(r) 23,000, 26,000, and 29,000 were specifically labeled with [alpha-32P]GTP in all the fractions examined and enriched in sarcolemmal membranes. The 23-kDa polypeptide was labeled to a higher extent with [alpha-32P]GTP than the 26- and 29-kDa polypeptides. A polypeptide of M(r) 40,000 was weakly labeled with [alpha-32P]GTP in the sarcolemmal membrane and tentatively identified as Gi alpha by immunostaining with anti-Gi alpha antibodies. Cytosolic GTP-binding proteins were labeled with [alpha-32P]GTP and their potential sites of interaction investigated using the blot overlay approach. A polypeptide of 32 kDa present in sarcolemmal membranes, intercalated discs, and enriched in heart gap junctions was identified as a major site of interaction. The low molecular weight GTP-binding proteins associated with the 32-kDa polypeptide through a complex involving cytosolic components of M(r) 56,000, 36,000, 26,000, 23,000, and 12,000. A monoclonal antibody against connexin 32 from liver strongly recognized the 32-kDa polypeptide in heart gap junctions, whereas polyclonal antibodies only weakly reacted with this polypeptide. The low molecular weight GTP-binding proteins associated with a 32-kDa polypeptide in liver membranes that was also immunologically related to connexin 32. These results indicate the presence of a subset of low molecular weight GTP-binding proteins in a membrane-associated and a cytoplasmic pool in cardiac muscle. Their association with a 32-kDa component that is related to the connexins suggests that these polypeptides may be uniquely situated to modulate communication at the cell membrane.     

Identification of low molecular weight GTP-binding proteins and their sites of interaction in subcellular fractions from skeletal muscle

The presence of low molecular weight GTP-binding proteins was investigated in subcellular fractions from skeletal muscle. Skeletal muscle homogenate, transverse tubules, triads, sarcoplasmic reticulum membranes, and cytosol fractions were separated in sodium dodecyl sulfate-gel electrophoresis and blotted onto nitrocellulose. The presence of GTP-binding proteins was explored by incubation of these blots with [alpha-32P] GTP. GTP labeled two polypeptides of Mr = 23,000 and 29,000 in all the fractions examined. Binding of [alpha-32P]GTP was specific and dependent on Mg2+. The 23-kDa polypeptide was labeled to a higher extent with [alpha-32P]GTP than the 29-kDa polypeptide, although both were enriched in transverse tubule fractions. A GTP-binding polypeptide of 40 kDa was also enriched in transverse tubule preparations and identified as Gi alpha by immunostaining with anti-Gi alpha. Using a blot overlay approach and [alpha-32P]GTP-labeled cytosolic components, several polypeptides were identified that interact with the 23- and 29-kDa GTP-binding proteins. Among these components were polypeptides of Mr = 60,000, 47,000, 44,000, 42,000, and 38,000, which were mainly of cytosolic origin but also associated with triads and transverse tubule membranes. The 47-, 44-, 42-, and 38-kDa polypeptides were found to be structurally related to the glycolytic enzymes enolase, 3-phosphoglyceric phosphokinase, aldolase, and glycoeraldehyde-3-phosphate dehydrogenase, respectively. The purified glycolytic enzymes specifically bound the 23- and 29-kDa GTP-binding proteins under both denaturing and nondenaturing conditions. The association of the GTP-binding proteins with these polypeptides was resistant to detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), Triton X-100, and Tween. A 23-kDa GTP-binding protein purified from chromaffin cells bound to a 157-kDa polypeptide in triads and chromaffin cell membranes. The 157-kDa polypeptide was a minor component in these membranes and not related to the subunits of the dihydropyridine receptor. In view of the proposed function of low molecular weight GTP-binding proteins in processes such as membrane communication and secretion coupling, the association of these proteins with transverse tubules and triads in skeletal muscle is discussed in terms of a role in signal transmission.     

Primary structure and possible origin of the non-glycosylated basic proline-rich protein of human submandibular/sublingual saliva.

As a first step in determining the molecular mechanism of membrane fusion stimulated by GTP in rough endoplasmic reticulum (RER), we have looked for GTP-binding proteins. Rough microsomes from rat liver were treated for the release of ribosomes, and the membrane proteins were separated by SDS/polyacrylamide-gel electrophoresis. The polypeptides were then blotted on to nitrocellulose sheets and incubated with [alpha-32P]GTP [Bhullar & Haslam (1987) Biochem. J. 245, 617-620]. A doublet of polypeptides (23 and 24 kDa) was detected in the presence of 2 microM-MgCl2. Binding of [alpha-32P]GTP was blocked by 1-5 mM-EDTA, 10-10,000 nM-GTP or 10 microM-GDP. Either guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta gamma-imido]triphosphate at 100 nM completely inhibited binding, but ATP, CTP or UTP at 10 mciroM did not. Pretreatment of microsomes by mild trypsin treatment (0.5-10 micrograms of trypsin/ml, concentrations known not to affect microsomal permeability) led to inhibition of [alpha-32P]GTP binding, suggesting a cytosolic membrane orientation for the GTP-binding proteins. Two-dimensional gel-electrophoretic analysis revealed the 23 and 24 kDa [alpha-32P]GTP-binding proteins to have similar acid isoelectric points. [alpha-32P]GTP binding occurred to similar proteins of rough microsomes from rat liver, rat prostate and dog pancreas, as well as to a 23 kDa protein of rough microsomes from frog liver, but occurred to distinctly different proteins in a rat liver plasma-membrane-enriched fraction. Thus [alpha-32P]GTP binding has been demonstrated to two low-molecular-mass (approx. 21 kDa) proteins in the rough endoplasmic reticulum of several varied cell types.     

Relationship among methylation,isoprenylation, and GTP binding in 21-to 23-kDa proteins of neuroblastoma

1. Dimethylsulfoxide-induced differentiated neuroblastoma express high levels of membrane 21 to 23-kDa carboxyl methylated proteins. Relationships among methylation, isoprenylation, and GTP binding in these proteins were investigated. Protein carboxyl methylation, protein isoprenylation, and [alpha-32P]GTP binding were determined in the electrophoretically separated proteins of cells labeled with the methylation precursor [methyl-3H]methionine or with an isoprenoid precursor [3H]mevalonate. 2. A broad band of GTP-binding proteins, which overlaps with the methylated 21 to 23-kDa proteins, was detected in [alpha-32P]GTP blot overlay assays. This band of proteins was separated in two-dimensional gels into nine methylated proteins, of which four bound GTP. 3. The carboxyl-methylated 21 to 23-kDa proteins incorporated [3H]mevalonate metabolites with characteristics of protein isoprenylation. The label was not removed by organic solvents or destroyed by hydroxylamine. Incorporation of radioactivity from [3H]mevalonate was enhanced when endogenous levels of mevalonate were reduced by lovastatin, an inhibitor of mevalonate synthesis. Lovastatin blocked methylation of the 21 to 23-kDa proteins as well (greater than 70%). 4. Methylthioadenosine, a methylation inhibitor, inhibited methylation of these proteins (greater than 80%) but did not affect their labeling by [3H]mevalonate. The results suggest that methylation of the 21 to 23-kDa proteins depends on, and is subsequent to, isoprenylation. The sequence of events may be similar to that known in ras proteins, i.e., carboxyl methylation of a C-terminal cysteine that is isoprenylated. 5. Lovastatin reduced the level of small GTP-binding proteins in the membranes and increased GTP binding in the cytosol. Methylthioadensoine blocked methylation without affecting GTP binding. 6. Thus, isoprenylation appears to precede methylation and to be important for membrane association, while methylation is not required for GTP binding or membrane association. The role of methylation remains to be determined but might be related to specific interactions of the small GTP-binding proteins with other proteins.     

Gn-proteins are distinct from ras p21 and other known low molecular mass GTP-binding proteins in the platelet

The 27 kDa platelet membrane protein (Gn27) that binds [alpha-32P]GTP on nitrocellulose blots of SDS-polyacrylamide gels [(1987) Biochem. J. 245, 617-620] was compared with other low molecular mass GTP-binding proteins. Platelet membranes also contained 21 kDa proteins that bound anti-ras p21 antibody and 22-23 kDa proteins that could be ADP-ribosylated by botulinum neurotoxin type D. These groups of proteins were resolved electrophoretically from each other and from Gn27. A low molecular mass GTP-binding protein from bovine brain [(1987) Biochem. J. 246, 431-439] was also resolved from Gn27. At the levels normally present in cell membranes, only Gn-proteins bound significant amounts of [32P]GTP after transfer of protein from SDS-polyacrylamide gels to nitrocellulose.     

Detection of 23-27 kDa GTP-binding proteins in platelets and other cells.

Membrane proteins from rabbit and human platelets were separated by SDS/polyacrylamide-gel electrophoresis and the resolved polypeptides blotted on nitrocellulose. A family of GTP-binding proteins, termed Gn proteins, was detected by incubation of these blots with [alpha-32P]GTP in the presence of Mg2+. A major Gn protein with a molecular mass of 27 kDa (Gn27) and lesser amounts of 23, 24 and 25 kDa Gn proteins were observed in platelet membranes; much smaller amounts were in the platelet soluble fraction. Binding of [alpha-32P]GTP by platelet Gn proteins was blocked by GDP, GTP or guanosine 5'-[gamma-thio]triphosphate, but not by GMP or adenosine 5'-[beta gamma-imido]triphosphate. Rabbit and human red-cell membranes contained only Gn27. When rat tissues were analysed for Gn proteins, the largest amounts were found in brain, which contained two membrane-bound forms (Gn27 and Gn26) and a soluble form (Gn26).     

Presence of guanine nucleotide-binding proteins in a plant hypocotyl microsomal fraction

The presence of specific guanine nucleotide-binding proteins in a zucchini (Cucurbita pepo L.) hypocotyl microsomal fraction was investigated. Polypeptides were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of nitrocellulose blots with [alpha-32P]GTP and [gamma-32P]GTP indicated the presence of four specific and distinct GTP-binding proteins with molecular masses of approx. 23.4 kDa, 24.8 kDa, 26.6 kDa and 28.5 kDa. Binding of [alpha-32P]GTP could be completely prevented by 30 microM GDP or 10 microM guanosine 5'[gamma-thio]triphosphate. This report presents evidence for the presence in a microsomal fraction from zucchini hypocotyls of Gn-proteins as defined by Bhullar and Haslam (1987) Biochem.J. 245, 617-620. The four plant proteins resemble animal Gn-proteins when molecular weights and GTP-binding specificities are considered.     

Localization of ras antigenicity in rat hepatocyte plasma membrane and rough endoplasmic reticulum fractions

We have examined the antigenicity of plasma membrane (PM) and rough microsomal (RM) fractions from rat liver using anti-ras monoclonal antibodies 142-24EO5 and Y13-259 and immunochemistry as well as electron microscope immunocytochemistry. Proteins immunoprecipitated with monoclonal antibody 142-24E05 were separated using single-dimensional gradient-gel electrophoresis. The separated proteins were then blotted onto nitrocellulose sheets and incubated with [alpha-32P]GTP. Radioautograms of blots indicated the presence of specific 21.5- and 22-kDa labeled proteins in the PM fraction. A 23.5-kDa [alpha-32P] GTP-binding protein was detected in immunoprecipitates of both PM and RM fractions. Monoclonal antibody Y13-259 reacted only with the 21.5-kDa [alpha-32P] GTP-binding protein in the plasma membrane fraction. When anti-ras monoclonal antibody 142-24E05 and the immunogold technique were applied to membrane fractions using a preembedding immunocytochemical method, specific labeling was observed in association with both vesicular structures and membrane sheets in the PM fraction but only with electron-dense vesicular structures in the RM fraction. Thus ras antigenicity is associated with hepatocyte plasma membranes and ras-like antigenicity is probably associated with vesicular (secretory/endocytic) elements in both plasma membrane and rough microsomal preparations.     

The mitotic apparatus-associated 51-kDa protein from sea urchin eggs is a GTP-binding protein and is immunologically related to yeast polypeptide elongation factor 1 alpha

We investigated the biochemical characteristics of the 51-kDa protein that is a major mitotic apparatus-associated basic protein of sea urchin eggs (Toriyama, M., Ohta, K., Endo, S., and Sakai, H. (1988) Cell Motil. Cytoskeleton 9, 117-128). The amino acid composition of the 51-kDa protein was apparently different from those of tubulin, actin, histones, and myelin basic protein; yet it was similar to those of polypeptide elongation factors 1 alpha (EF-1 alpha). In addition, antibody to EF-1 alpha from yeast cross-reacted with the 51-kDa protein. [3H] GTP binding activity was detected in the phosphocellulose-purified fraction (PC fraction) which predominantly contained the 51-kDa protein and was shown to be specific to GTP, GDP, guanylyl imidodiphosphate, and ITP. Photo-affinity labeling using [alpha-32P]8-azidoguanosine triphosphate (8-azido-GTP) demonstrated that a 51-kDa polypeptide in the PC fraction specifically bound 8-azido-GTP. This GTP-binding polypeptide was bound to a GTP affinity column, could be eluted by the addition of GTP, and was immunoreactive with anti-51-kDa protein antibodies. When the PC fraction was applied to a gel filtration chromatography column, GTP binding activity was completely coeluted with the 51-kDa protein. Furthermore, the PC fraction and the gel filtration-purified fraction had EF-1 alpha activity: [14C]Phe-tRNA transferring activity to ribosomes in the presence of poly(U) and ribosome-dependent GTPase activity. The results indicate that the mitotic apparatus-associated 51-kDa protein is a GTP-binding protein and suggest that it is structurally and functionally related to yeast EF-1 alpha.     

A novel small molecular weight GTP-binding protein with the same putative effector domain as the ras proteins in bovine brain membranes. Purification, determination of primary structure, and characterization

In the present studies, we have purified a novel small Mr GTP-binding protein, designated as smg p21, to near homogeneity from bovine brain crude membranes, isolated the complementary DNA (cDNA) of this protein from a bovine brain cDNA library, determined the complete nucleotide and deduced amino acid sequences, and characterized the kinetic properties. The cDNA of smg p21 has an open reading frame encoding a protein of 184 amino acids with a calculated Mr of 20,987. The Mr of purified smg p21 is estimated to be about 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Homology search indicates that smg p21 is a novel protein with the consensus amino acid sequences for GTP/GDP-binding and GTPase domains but shares about 55% amino acid sequence homology with the human c-Ha-ras protein. Moreover, smg p21 has the same putative effector domain as the Ha-, Ki-, and N-ras proteins at the same position and the same consensus C-terminal sequence as in these ras proteins. Consistent with these structural properties, smg p21 binds specifically [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), GTP, and GDP with a Kd value for GTP gamma S of about 40 nM. smg p21 binds about 0.7 mol of GTP gamma S/mol of protein. [35S]GTP gamma S-binding to smg p21 is inhibited by pretreatment with N-ethylmaleimide.smg p21 hydrolyzes GTP to liberate Pi with a turnover number of about 0.007 min-1. These kinetic properties of smg p21 are similar to those of the c-ras proteins. These results suggest that smg p21 is a novel GTP-binding protein exerting action(s) similar or antagonistic to that (those) of the ras proteins.     

Identification of multiple ral gene products in human platelets that account for some but not all of the platelet Gn-proteins

Polyclonal antibodies raised against specific recombinant low molecular mass GTP-binding proteins were tested for their ability to recognize partially purified human platelet membrane Gn-proteins (i.e. proteins that bind [alpha-32P]GTP on nitrocellulose blots of SDS/polyacrylamide gels). An antiserum against simian ralA protein recognized a 27 kDa human platelet protein with the same apparent molecular mass as the major platelet Gn-protein (Gn27). In further analysis by two-dimensional polyacrylamide gel electrophoresis, the isoelectric focusing step permitted resolution of 12 major Gn-protein forms, seven of 27 kDa (Gn27a-g), one of 26 kDa (Gn26) and four of 24 kDa (Gn24a-d). The ralA antibody reacted strongly with the five most basic Gn27 species (a-e), weakly with Gn26 and not at all with Gn27f, Gn27g or Gn24a-d. We conclude that ral gene products account for some but probably not for all of the platelet Gn-proteins.     

Phospholipase C activity in NCB-20 cells is inhibited by protein kinase A-mediated phosphorylation of low molecular mass GTP-binding proteins.

We have previously shown that bradykinin-induced production of second messengers such as inositol trisphosphate and diacylglycerol in neurotumor cells is inhibited by raising cellular cyclic AMP levels, which in turn inhibit phospholipase C. A monoclonal antibody to phospholipase C-II immunoprecipitated the 140-kDa form of phospholipase C-II from [35S]methionine/[3H]eucine-labeled cells, but not [32P]orthophosphate-labeled phospholipase C-II, following treatment with either forskolin or dibutyryl cyclic AMP. This suggested that phospholipase C is not the target for cyclic AMP-dependent protein kinase-mediated phosphorylation. In vitro studies confirmed that phospholipase C activity was inhibited by raising cellular cAMP levels, and partial sensitivity to Bordetella pertussis toxin suggested the involvement of a GTP-binding protein which could be the target for protein kinase A. The involvement of a GTP-binding protein in coupling the bradykinin receptor to phospholipase C was further suggested by the ability of both guanosine 5'-O-(thio-triphosphate) and fluoride (NaF) to release inositol phosphates from NCB-20 cell membranes previously labeled with [3H]inositol. Both effects were blocked by pretreatment of the cells with protein kinase A activators, further suggesting a GTP-binding protein as the target for protein kinase A-mediated phosphorylation. When whole NCB-20 cell extracts were blotted onto nitrocellulose and incubated with [alpha- 32P]GTP, a major 24-kDa band plus minor bands at 22 and 20 kDa were revealed by autoradiography. A pH 3.0/6.0 soluble (basic protein) NCB-20 cell extract revealed the major 24-kDa band plus the 20-kDa band, and similar basic proteins were shown to be heavily phosphorylated following [32P]orthophosphate labeling and pretreatment with forskolin. The size and ability to bind GTP on Western blots are characteristic of the ras, rho, smg, etc. family of GTP-binding proteins recently suggested to be the much sought after GPLC (Lapetina, E.G., Lacal, J. C., Reep, B. R., and Molina y Vedia, L. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3131-3134; Wang, P., Nishihata, J., Takabori, E., Yamamoto, K., Toyoshima, S., and Osawa, T. (1989) J. Biochem. (Tokyo) 105, 461-466; Nagata, K.-I., Nagao, S., and Nozawa, Y. (1989) Biochem. Biophys. Res. Commun. 160, 235-242). We propose that GPLC is uniquely sensitive to protein kinase A-mediated phosphorylation and that phosphorylation inhibits stimulus-secretion coupling in these cells.     

Molecular cloning and expression of a G25K cDNA, the human homolog of the yeast cell cycle gene CDC42.

G25K is a low-molecular-mass GTP-binding protein with a broad distribution in mammalian tissues. A cDNA clone was isolated by using oligonucleotides corresponding to the partial amino acid sequence of purified human G25K. The cDNA encodes an 191-amino-acid polypeptide containing GTP-binding consensus sequences and a putative farnesylation site at the C terminus. The sequence exhibits 50 and 70% identities to the mammalian rho and rac proteins, respectively, and an 80% identity to the Saccharomyces cerevisiae CDC42 gene product. Insect Sf9 cells infected with recombinant baculovirus vectors expressing the G25K cDNA produced a 25-kDa protein that bound GTP and was recognized by antibodies specifically reactive to G25K. G25K appears to be the human homolog of the CDC42 gene product, since expression of the G25K cDNA in S. cerevisiae suppressed both cdc42-1 and cdc24-4 temperature-sensitive lethal mutations.     

Isoprenylation of the low molecular mass GTP-binding proteins rac 1 and rac 2: possible role in membrane localization

Ras proteins can be modified at their COOH-terminal cysteine in the motif Cys-Ali-Ali-Xaa by a farnesyl isoprenoid. This modification is essential for membrane association and biological activity of ras proteins. A similar COOH-terminal amino acid sequence, Cys-Xaa-Ali-Xaa, exists in the ras-related GTP-binding proteins rac 1 and rac 2. To determine whether these proteins were similarly modified, COS cells were transfected with rac 1 and rac 2 cDNA and expressed proteins were labeled with [3H]mevalonic acid. We report here that both rac 1 and rac 2 are post-translationally modified by addition of an isoprenoid group, the likely site of which is the COOH-terminal cysteine. Isoprenylation was found only in racs associated with particulate cell fractions, suggesting that this modification may be associated with membrane localization of the proteins. These data specifically identify mammalian low molecular mass GTP-binding proteins other than ras that undergo post-translational modification and further define the COOH-terminal consensus sequence, Cys-Ali-Ali-Xaa, as an isoprenylation signal. This sequence may identify a larger family of low molecular mass GTP-binding proteins which are isoprenylated.     

The identification and characterization of an epidermal growth factor-stimulated phosphorylation of a specific low molecular weight GTP-binding protein in a reconstituted phospholipid vesicle system

The abilities of different GTP-binding proteins to serve as phosphosubstrates for the epidermal growth factor (EGF) receptor/tyrosine kinase have been examined in reconstituted phospholipid vesicle systems. During the course of these studies we discovered that a low molecular mass, high affinity GTP-binding protein from bovine brain (designated as the 22-kDa protein) served as an excellent phosphosubstrate for the tyrosine-agarose-purified human placental EGF receptor. The EGF-stimulated phosphorylation of the purified 22-kDa protein occurs on tyrosine residues, with stoichiometries approaching 2 mol of 32Pi incorporated/mol of [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-binding sites. The EGF-stimulated phosphorylation of the brain 22-kDa protein requires its reconstitution into phospholipid vesicles. No phosphorylation of this GTP-binding protein is detected if it is simply mixed with the purified EGF receptor in detergent solution or if detergent is added back to lipid vesicles containing the EGF receptor and the 22-kDa protein. The EGF-stimulated phosphorylation of this GTP-binding protein is also markedly attenuated by guanine nucleotides, i.e. GTP, GTP gamma S, or GDP, suggesting that maximal phosphorylation occurs when the GTP-binding protein is in a guanine nucleotide-depleted state. Purified preparations of the 22-kDa phosphosubstrate do not cross-react with antibodies against the ras proteins. However, they do cross-react against two different peptide antibodies generated against specific sequences of the human platelet (and placental) GTP-binding protein originally designated Gp (Evans, T., Brown, M. L., Fraser, E. D., and Northrup, J. K. (1986) J. Biol. Chem. 261, 7052-7059) and more recently named G25K (Polakis, P. G., Synderman, R., and Evans, T. (1989) Biochem. Biophys. Res. Commun. 160, 25-32). When highly purified preparations of the human platelet Gp (G25K) protein are reconstituted with the purified EGF receptor into phospholipid vesicles, an EGF-stimulated phosphorylation of the platelet GTP-binding protein occurs with a stoichiometry approaching 2 mol of 32Pi incorporated/mol of [35S]GTP gamma S-binding sites. As is the case for the brain 22-kDa protein, the EGF-stimulated phosphorylation of the platelet GTP-binding protein is attenuated by guanine nucleotides. Overall, these results suggest that the brain 22-kDa phosphosubstrate for the EGF receptor is very similar, if not identical, to the Gp (G25K) protein. Although guanine nucleotide binding to the brain 22-kDa protein or to the platelet. GTP-binding protein inhibits phosphorylation, the phosphorylated GTP-binding proteins appear to bind [35S]GTP gamma S slightly better than their nonphosphorylated counterparts.     

Immunodetection of ralA and ralB GTP-binding proteins in various rat tissues and platelets

Polyclonal antibodies were generated against a synthetic peptide corresponding to the C-terminal (amino acids 192-204) region of ralA and ralB GTP-binding proteins. The ralA and ralB antibodies recognized a 27 kDa protein in the human platelet particulate fraction. Incubation of ralA antibodies with ralB immunizing peptide and ralB antibodies with ralA immunizing peptide prior to Western blotting did not abolish the ability of antibodies to recognize the 27 kDa protein in human platelet particulate fraction. However, when antibodies were incubated with the respective immunizing peptide prior to Western blotting, the 27 kDa human platelet protein was no longer recognized by the antibodies. Incubation of nitrocellulose blots containing polypeptides separated using SDS-PAGE with [-³²P]GTP demonstrated the presence of GTP-binding proteins of molecular mass between 23-27 kDa in rat platelets and the various tissues tested. Analysis using subtype specific antibodies demonstrated that both ralA and ralB GTP-binding proteins were expressed in rat platelets and the various tissues tested. The protein recognized by the ralA and ralB antibodies in rat tissues and platelets had mobility on SDS-PAGE identical to that of the human platelet ral protein. Varying amounts of these proteins were detected in all the tissues tested except white muscle which contained very low level of ralB protein. The widespread distribution of ralA and ralB GTP-binding proteins suggests that they may participate in a common pathway in mammalian cells and tissues.     

Isoprenylation and carboxylmethylation in small GTP-binding proteins of pheochromocytoma (PC-12) cells

1. A group of 21 to 24-kDa proteins of pheochromocytoma (PC-12) cells was found in blot overlay assays to bind specifically [alpha-32P]GTP. Binding was inhibited by GTP analogues but not by ATP. Such small GTP-binding proteins were found in the cytosolic and in the particulate fraction of the cells, but they were unevenly distributed: about 75% of the small GTP-binding proteins were localized within the particulate fraction of the cells. Separation of these proteins by two-dimensional gel electrophoresis revealed the existence of seven distinct [alpha-32P]GTP-binding proteins. 2. Targeting of the small GTP-binding proteins to the particulate fraction of PC-12 cells requires modification by isoprenoids, since depleting the cells of the isoprenoid precursor mevalonic acid (MVA) by the use of lovastatin resulted in a 50% decrease in membrane-bound small GTP-binding proteins, with a proportionate increase in the cytosolic form. This blocking effect of lovastatin was reversed by exogenously added MVA. 3. In addition, metabolic labeling of PC-12 cells with [3H]MVA revealed incorporation of [3H]MVA metabolites into the cluster of 21 to 24-kDa proteins in a form typical of isoprenoids; the label was not removed from the proteins by hydroxylamine, and labeling was enhanced in cells incubated with lovastatin. The latter effect reflects a decrease in the isotopic dilution of the exogenously added [3H]MVA, as the addition of exogenous MVA reversed the effect of lovastatin on [3H]MVA-metabolite incorporation into the 21 to 24-kDa proteins. 4. Additional experiments demonstrated that isoprenylation is required not only for membrane association of small GTP-binding proteins, but also for their further modification by a methylation enzyme. This was evident in experiments in which the cells were metabolically labeled with [methyl-3H]methionine, a methylation precursor. The group of 21 to 24-kDa proteins was labeled with a methyl-3H group in a form typical of C-terminal-cysteinyl carboxylmethyl esters. Their methylation was blocked by the methylation inhibitors methylthioadenosine (MTA), 3-deazadenosine and homocysteine thiolactone as well as by lovastatin. MVA reversed the lovastatin block of methylation. 5. Two-dimensional gel analysis of the [3H]methylated proteins detected seven methylated small GTP-binding proteins that correspond to the isoprenylated proteins. Levels of the small GTP-binding proteins as well as isoprenylation and methylation were reduced by cycloheximide. 6. Distribution of the methylated proteins between particulate and cytosolic fractions was found to be similar to that of the small GTP-binding proteins (i.e., a 4:1 ratio).(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification, identification, and characterization of two GTP-binding proteins with molecular weights of 25,000 and 21,000 in human platelet cytosol. One is the rap1/smg21/Krev-1 protein and the other is a novel GTP-binding protein

We have purified, characterized, and identified two GTP-binding proteins with Mr of 25,000 (c25KG) and 21,000 (c21KG) from the cytosol fraction of human platelets. These two proteins were not copurified with the beta gamma subunits of heterotrimeric GTP-binding proteins. Amino acid sequences of tryptic fragments of c21KG completely matched with those of rap1 protein (Pizon, V., Chardin, P., Lerosey, I., Olofsson, B., and Tavitian, A. (1988) Oncogene 3, 201-204), smg p21 (Kawata, M., Matsui, Y., Kondo, J., Hishida, T., Teranishi, Y., and Takai, Y. (1988) J. Biol. Chem. 263, 18965-18971), and Krev-1 protein (Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y., and Noda, M. (1989) Cell 56, 77-84). The partial amino acid sequence analysis of c25KG revealed that this protein was different from any low Mr GTP-binding proteins already reported. c25KG bound about 1 mol of [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein, with a Kd value of about 45 nM. [35S]GTP gamma S-binding to c25KG was specifically inhibited by guanine nucleotides, GTP and GDP, but not by adenine nucleotides such as ATP and adenyl-5'-yl beta, gamma-imidodiphosphate. The binding activity was not inhibited by pretreatment with N-ethylmaleimide. c25KG hydrolyzed GTP to librate Pi with the specific activity of 1.8 mmol of Pi/mol of protein/min, which are different from the activities of the already purified low Mr GTP-binding proteins. We conclude that c25KG is a novel GTP-binding protein and c21KG is a rap1/smg p21/Krev-1 product.