Abstract
Cells are capable of responding to extracellular stimuli because of their ability to detect external information and to transduce this signal to intracellular effectors that may result in the generation of second messengers and/or cause alteration in the cell’s metabolism. The cell surface receptors are responsible for detecting the external information and a group of proteins, termed GTP-binding proteins, are responsible for receptor-effector coupling. In eukaryotic cells, there are two major families of GTP-binding proteins (G proteins) that participate in the various signal transduction pathways (Bourne et al., 1990, 1991). The first family consists of the heterotrimeric (αβγ-subunit structure) GTP-binding proteins, which link cell surface receptors to effectors inside the cell (Birnbaumer et al., 1990; Neer, 1995; Rens-Domiano and Hamm, 1995). The second family includes GTP-binding proteins that consist of a single polypeptide chain and are commonly referred to as the small-mol-wt G proteins (Grand and Owen, 1991; Valencia et al., 1991; Takai et al., 1992; Bokoch and Der, 1993). This chapter will deal with the characterization and possible function(s) of small-mol-wt G proteins in mammalian cells.
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Alberts, A. W. (1988) Discovery, biochemistry and biology of lovastatin. Am. J. Physiol 62, 10J–15J.
Barbacid, M. (1987) Ras genes Ann Rev. Biochem. 56, 799–827
Barfod, E T., Zheng, Y, Kuang, W-J., Hart, M. J, Evans, T., Cerione, R. A and Ashkenazi, A. (1993) Cloning and expression of a human CDC42 GTPase-activating protein reveals a functional SH3-binding domain J. Biol. Chem. 268, 26,059–26,062.
Basu, T. N., Gutmann, D H., Fletcher, J A., Glover, T. W, Collins, E S. and Downward, J. (1992) Aberrant regulation of ratproteins in malignant tumour cells from type 1 neurofibromatosis patients. Nature (Lond.) 356, 713–715.
Bhullar, R. P. and Haslam, R J. (1987) Detection of 23–27 kDa GTP-binding proteins in platelets and other cells Biochem J 245, 617–620.
Bhullar, R P. (1992) Identification of some of the brain Gn27 as the ral gene product, comparison between the brain and platelet Gn proteins. FEBS Lett. 298, 61–64
Bhullar, R. P (1996) Expression of a 24 kDa GTP-binding protein (Gn24) is increased in lovastatin treated human erythroleukemia cells Mol Cell. Biochem. 156, 59–67.
Bhullar, R. P. and Seneviratne, H. D (1996) Characterization of human platelet GTPase activating protein for the ral GTP-binding protein Biochim. Biophys. Acta 1311, 181–188.
Bielinski, D. F., Pyun, H.-Y, Linko-Stentz, K., Macara, I. G., and Fine, R. E. (1993) Ral and rab3A are major GTP-binding proteins of axonal rapid transport and synaptic vesicles and do not redistribute following depolarization stimulated synaptosomal exocytosis. Biochim. Biophys. Acta 1151, 246–256
Birnbaumer, L., Abramowitz, J., and Brown, A. M. (1990) Receptor-effector coupling by G proteins. Biochim. Biophys. Acta 1031, 163–224.
Bischoff, F. R. and Ponstingi, H. (1991) Mitotic regulator protein RCCl is complexed with a nuclear ras-related polypeptide. Proc. Natl. Acad. Sci. USA 88, 10,830–10,834.
Boguski, M S. and McCormick, F. (1993) Proteins regulating ras and its relatives. Nature (Lond ) 366, 643–654.
Bokoch, G. M. and Der, C. J. (1993) Emerging concepts in the ras super-family of GTP-binding proteins FASEB J. 7, 750–759.
Boman, A. L. and Kahn, R. A. (1995) Arf proteins: the membrane traffic police? Trends Biochem. Sci. 20, 147–150.
Bourne, H. R., Sanders, D. A., and McCormick, F. (1990) The GTPase superfamily. conserved structure and molecular mechanism. Nature (Lond.) 348, 125–132.
Bourne, H. R., Sanders, D. A., and McCormick, F. (1991) The GTPase superfamily a conserved switch for diverse cell functions. Nature (Lond.) 349, 117–127.
Brown, H. A., Gutowski, S., Moomaw, C. R., Slaughter, C, and Sternweis, P. C. (1993) ADP-nbosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity. Cell 75, 1137–1144.
Casey, P. J., Solski, P A., Der, C. J., and Buss, J. E. (1989) p21ras is modified by a farnesyl isoprenoid. Proc. Natl Acad Sci. USA 86, 8323–8327
Casey, P. J. (1995) Protein lipidation in cell signaling. Science 268, 221–225.
Chardin, P. (1988) The ras superfamily proteins Biochimie 70, 865–868
Chardin, P. and Tavitian, A. (1986) The ral gene: a new ras related gene isolated by the use of a synthetic probe. EMBO J. 9, 2203–2208.
Chardin, P, Madaule, P, and Tavitian, A. (1988) Coding sequence of human rho cDNAs clone 6 and clone 9. Nucleic Acids Res. 16, 2717
Chardin, P. and Tavitian, A. (1989) The coding sequence of human ralA and ralB. Nucleic Acids Res. 17, 4380.
Chavner, P., Vingron, M., Sander, C, Simons, K. and Zerial, M (1990) Molecular cloning of YPT1/SEC4-related cDNAs from an epithelial cell line. Mol. Cell. Biol. 10, 6578–6585.
Chavrier, P., Simons, K., and Zerial, M. (1992) The complexity of the Rab and Rho GTP-binding protein subfamilies revealed by a PCR cloning approach Gene 112, 261–264.
Clark, J., Moore, L., Krasinskas, A., Way, J., Battey, J., Tamkun, J., and Kahn, R A (1993) Selective amplification of additional members of the ADP-ribosylation factor (ARF) family cloning of additional human and Drosophila ARF-like genes. Proc. Natl. Acad. Sci. USA 90, 8952–8956.
Comerford, I. G., Gibson, J. R., Dawson, A. P., and Gibson, I. (1989) Ras p21 and other Gn proteins are detected in mammalian cell lines by [gamma-35S]GTP gamma S binding. Biochem Biophys. Res. Comm 159, 1269–1274.
Didsbury, J., Weber, R. F., Bokoch, G. M., Evans, T., and Snyderman, R. (1989) rac a novel ras-related family of proteins that are botulinum toxin substrates. J. Biol. Chem. 264, 16,378–16,382.
Drivas, G. T., Shih, A., Coutavas, E, Rush, M. G., and D’Eustachio, P. (1990) Characterization of four novel ras-like genes expressed in a human teratocarcinoma cell line Mol. Cell. Biol. 10, 1793–1798.
Egan, S. E., Giddings, B. W., Brooks, M. W., Buday, L., Sizeland, A M., and Weinberg, R. A. (1993) Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation Nature (Lond.) 363, 45–51
Emkey, R., Freedman, S., and Feig, L. (1991) Characterization of a GTPase-activating protein for the ras-related ral protein. J. Biol. Chem. 266, 9703–9706.
Evans, T, Brown, M. L., Fraser, E. D., and Northup, J. K. (1986) Purification of the major GTP-binding proteins from human placental membranes. J. Biol Chem. 261, 7052–7059.
Farnsworth, C C, Seabra, M. C, Ericsson, L. H, Gelb, M. H., and Glomset, J. A. (1994) Rab geranylgeranyl transferase catalyzes the geranylgeranylation of adjacent cysteines in the small GTPases Rab1A, Rab3A, and Rab5A. Proc. Natl. Acad Sci. USA 91, 11,963–11,967.
Farrell, F. X., Yamamoto, K., and Lapetina, E G (1993) Prenyl group identification of rap2 proteins: a ras superfamily member other than ras that is farnesylated. Biochem. J. 289, 349–355.
Fischer von Mollard, G., Mignery, G. A., Baumert, M, Perin, M. S., Hanson, T. J., Burger, P. M., Jahn, R., and Sudhof, T. C. (1990) rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles Proc. Natl. Acad Sci. USA 87, 1988–1992.
Fischer von Mollard, G., Sudhof, T. C, and Jahn, R. (1991) A small GTP-binding protein dissociates from synaptic vesicles during exocytosis. Nature (Lond.) 349, 79–81.
Fischer von Mollard, G., Stahl, B, Khokhlatchev, A., Sudhof, T. C, and Jahn, R. (1994) Rab3C is a synaptic vesicle protein that dissociates from synaptic vesicles after stimulation of exocytosis. J. Biol Chem. 269, 10,971–10,974.
Garrett, M. D., Major, G. N., Totty, N., and Hall, A. (1991) Purification and N-terminal sequence of the p21rho GTPase-activating protein, rho GAP. Biochem J. 276, 833–836.
Geppert, M, Bolshakov, V. Y., Siegelbaum, S. A., Takei, K., De-Camilli, P., Hammer, R. E., and Sudhof, T. C. (1994) The role of Rab3A in neuro-transmitter release. Nature (Lond.) 369, 493–497.
Gibbs, J. B, Schaber, M. D., Allard, W. J., Sigal, I S, and Scolnick, E. M (1988) Purification of ras GTPase activating protein from bovine brain. Proc. Natl. Acad. Sci. USA 85, 5026–5030.
Grand, R. J. and Owen, D. (1991) The biochemistry of ras p21. Biochem. J. 279, 635–640.
Hancock, J. F., Magee, A. L, Childs, J. E., and Marshall, C. J. (1989) All ras proteins are polyisoprenylated but only some are palmitoylated Cell 57, 1167–1177.
Hattori, S., Ohmi, N, Maekawa, M., Hoshino, M., Kawakita, M., and Nakamura, S. (1991) Antibody against neurofibromatosis type 1 gene product reacts with a triton-insoluble GTPase activating protein toward ras p21. Biochem. Biophys. Res. Commun. 177, 83–89.
Haubruck, H, Disela, C, Wagner, P. and Gallwitz, D. (1987) The ras related ypt protein is an ubiquitous eukaryotic protein: isolation and sequence analysis of mouse cDNA clones highly homologous to the yeast YPT1 gene EMBO J. 6, 4049–4053.
Huber, L. A., Ullrich, O., Takai, Y., Lutcke, A., Dupree, P., Olkkonen, V., Virta, H., Hoop, M. J., Alexander, K., Peter, M., Zerial, M., and Simons, K. (1994) Mapping of Ras-related GTP-binding proteins by GTP overlay following two-dimensional gel electrophoresis. Proc Natl. Acad. Sci. USA 91, 7874–7878.
Jilkina, O. and Bhullar, R P (1996) Generation of antibodies specific for the ralA and ralB GTP-binding proteins and determination of their concentration and distribution in human platelets. Biochim. Biophys. Acta, 1314, 157–166.
Kahn, R A. and Gilman, A. G (1984) Purification of a protein cofactor required for ADP ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin. J. Biol. Chem 259, 6228–6234.
Kahn, R. A., Kern, F. G., Clark, J., Gelmann, E. P., and Rulka, C. (1991) Human ADP-ribosylation factors. A functionally conserved family of GTP-binding proteins. J. Biol. Chem. 266, 2606–2614.
Kawata, M., Matsui, Y., Kondo, J., Hishida, T., Teranishi, Y., and Takai, Y. (1988) 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 J. Biol. Chem. 263, 18,965–18,971.
Kikuchi, A., Sasaki, T., Araki, S., Hata, Y, and Takai, Y. (1989) Purification and characterization from bovine brain cytosol of two GTPase-activating proteins specific for smg p21, a GTP-binding protein having the same effector domain as c-ras p21s. J. Biol. Chem. 264, 9133–9136.
Kinsella, B. T., Erdman, R A. and Maltese, W A. (1991) Carboxyl-terminal isoprenylation of ras-related GTP-binding proteins encoded by racl, rac2, and ralA. J. Biol Chem. 266, 9786–9794.
Laemmli, U. K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature (Lond.) 227, 680–685
Leevers, S. J., Paterson, H. E, and Marshall, C. J. (1994) Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature (Lond ) 369, 411–414.
Leung, T., Manser, E, Tan, L., and Lim, L. (1995) A novel serine/threomne kinase binding the ras-related rhoA GTPase which translocates the kinase to peripheral membranes. J. Biol. Chem 270, 29,051–29,054.
Li, C, Takei, K., Geppert, M., Daniell, L., Stenius, K., Chapman, E. R., Jahn, R., De-Camilli, P., and Sudhof, T. C. (1994) Synaptic targeting of rabphilin-3A, a synaptic vesicle Ca2+/phospholipid-brnding protein, depends on rab3A/3C. Neuron 13, 885–898
Lledo, P. M., Johannes, L., Vernier, P., Zorec, R., Darchen, F, Vincent, J. D., Henry, J. P. and Mason, W. T. (1994) Rab3 proteins, key players in the control of exocytosis. Trends Neurosci. 17, 426–432
Lowe, D. G., Capon, D. J., Delwart, E., Sakagutchi, A. Y., Naylor, S. L., and Goeddel, D. V (1987) Structure of the human and murine R-ras genes, novel genes closely related to ras proto-oncogenes. Cell 48, 137–146.
Maltese, W. A. and Sheridan, K M (1990) Isoprenoid modification of G25K (Gp), a low molecular mass GTP-binding protein distinct from p21ras. J. Biol Chem. 265, 17,883–17,890.
Manser, E., Leung, T., Salihuddin, H., Tan, L., and Lim, L. (1993) A nonreceptor tyrosine kinase that inhibits the GTPase activity of p21cdc42. Nature (Lond.) 363, 364–367.
Manser, E., Leung, T, Salihuddin, H., Zhao, Z.-S, and Lim, L. (1994) A brain serine/threonine protein kinase activated by Cdc42 and Racl. Nature (Lond.) 367, 40–46.
Manser, E, Leung, T., and Lim, L. (1995) Identification of GTPase-activating proteins by nitrocellulose overlay assay, in Methods in Enzymology, vol. 256 (Balch, W. E., Der, C. J, and Hall, A, eds.), Academic, San Diego, pp. 130–139.
Marshall, M. S., Hill, W S., Ng, A. S., Vogel, U. S., Schaber, M D., Scolnick, E. M., Dixon, R. A., Sigal, I S., and Gibbs, J B. (1989) A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity EMBOJ 8, 1105–1110.
Marshall, M. S (1994) Ras target proteins in eukaryotic cells FASEB J 9, 1311–1318
Mizoguchi, A, Yano, Y, Hamaguchi, H., Yanagida, H., Ide, C., Zahraoui, A., Shirataki, H., Sasaki, T., and Takai, Y (1994) Localization of Rabphilin-3A on the synaptic vesicle. Biochem. Biophys. Res Comm. 202, 1235–1243.
Munemitsu, S., Innis, M. A, Clark, R., McCormick, F., Ullrich, O., and Polakis, P (1990) Molecular cloning and expression of a G25K cDNA, the human homolog of the yeast cell cycle gene CDC42. Mol. Cell. Biol. 10, 5977–5982.
Nagata, K., Satoh, T., Itoh, H., Kozawa, T., Okano, Y., Doi, T., Kaziro, Y, and Nozawa, Y. (1990) The ram: a novel low molecular weight GTP-binding protein cDNA from a rat megakaryocyte library. FEBS Lett. 275, 29–32.
Neer, E. J. (1995) Heterotrimeric G proteins: organizers of transmembrane signals. Cell 80, 249–257.
Nelson, T. J., Yoshioka, T., Toyoshima, S., Han, Y. F., and Alkon, D. L. (1994) Characterization of a GTP-binding protein implicated in both memory storage and interorganelle vesicle transport. Proc. Natl. Acad. Sci USA 91, 9287–9291.
Ngsee, J. K., Elferink, L A and Scheller, R. H. (1991) A family of ras-like GTP-binding proteins expressed in electromotor neurons. J. Biol Chem. 266, 2675–2680.
Nice, E C, Fabri, L., Hammacher, A., Holden, J., Simpson, R. J., and Burgess, A. W. (1992) The purification of a Rapl GTPase-activating protein from bovine brain cytosol. J. Biol. Chem. 267, 1546–1553.
Oberhauser, A. F., Monck, J. R., Balch, W. E., and Fernandez, J M (1992) Exocytotic fusion is activated by Rab3a peptides. Nature (Lond ) 360, 270–273.
O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J Biol. Chem. 250, 4007–4021.
Omer, C. A. and Gibbs, J. B. (1994) Protein prenylation in eukaryotic microorganisms: genetics, biology and biochemistry. Mol. Microbiol. 11, 219–225
Pizon, V., Chardin, P, Lerosey, I, Oloffsson, B, and Tavitian, A. (1988a) Human cDNAs rapt and rap2 homologous to the Drosophila gene DMS3 encode proteins closely related to ras in the effecter region. Oncogene 3, 201–204.
Pizon, V., Lerosey, L., Chardin, P., and Tavitian, A. (1988b) Nucleotide sequence of a human cDNA encoding a ras-related protein (rap1B). Nucleic Acids Res. 16, 7719.
Quilliam, L. A., Khosravi-Far, R., Huff, S. Y. and Der, C. J. (1995) Guanine nucleotide exchange factors: activators of the ras superfamily of proteins. Bioessays 17, 395–404.
Rens-Domiano, S. and Hamm, H. E. (1995) Structural and functional relationships of heterotrimeric G proteins. FASEB J. 9, 1059–1066.
Rothman, J. E. and Orci, L. (1992) Molecular dissection of the secretory pathway. Nature (Lond ) 355, 409–415.
Schafer, W R. and Rine, J. (1992) Protein prenylation genes, enzymes, targets, and functions Ann Rev Genet 30, 209–237
Shirataki, H., Kaibuchi, T., Yamaguchi, K., Wada, H., Horiuchi, H., and Takai, Y (1992) A possible target protein for smg-25A/rab3A small GTP-binding protein. J. Biol. Chem. 267, 10,946–10,949.
Shirataki, H., Kaibuchi, K., Sakoda, T., Kishida, S., Yamaguchi, T., Wada, K., Miyazaki, M., and Takai, Y. (1993) Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin. Mol Cell Biol. 13, 2061–2068.
Siddiqi, A. R., Smith, J. L., Ross, A. H, Qiu, R G, Symons, M, and Exton, J. H. (1995) Regulation of phospholipase D in HL60 cells. Evidence for a cytosolic phospholipase D. J. Biol. Chem. 270, 8466–8473
Sudhof, T C (1995) The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature (Lond.) 375, 645–653.
Takahashi, K., Sasaki, T., and Takai, Y. (1996) Heterodimer formation of prenylated rab3A with two rabphilin-3A molecules Biochem Biophys Res. Comm. 217, 979–986.
Takai, Y., Kaibuchi, K, Kikuchi, A, and Kawata, M. (1992) Small GTP-binding proteins. Int Rev. Cyt 133, 187–230.
Towbin, H., Staehlin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets, procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354
Trahey, M. and McCormick, F. (1987) Acytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants Science 238, 542–545
Tsuchiya, M., Price, S. R., Tsai, S C, Moss, J, and Vaughan, M (1991) Molecular identification of ADP-ribosylation factor mRNAs and their expression in mammalian cells. J. Biol. Chem 266, 2772–2777
Ullrich, O., Stenmark, H., Alexander, K., Huber, L. A., Kaibuchi, K., Sasaki, T., Takai, Y., and Zerial, M. (1993) Rab GDP dissociation inhibitor as a general regulator for the membrane association of ras protems. J. Biol. Chem. 268, 18,143–18,150.
Valencia, A., Chardin, P., Wittinghofer, A., and Sander, C. (1991) The ras protein family: evolutionary tree and role of conserved amino acids. Biochemistry 30, 4637–4648.
Vielh, E., Touchot, N., Zahraoui, A., and Tavitian, A. (1989) Nucleotide sequence of a rat cDNA: rab1B, encoding a rab1-YPT related protein Nucleic Acids Res. 17, 1770.
Vogel, U. S., Dixon, R. A., Schaber, M D, Diehl, R. E., Marshall, M. S., Scol-mick, E. M., Sigal, I. S. and Gibbs, J. B. (1989) Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature (Lond.) 335, 90–93.
Waldo, G. L, Evans, T., Fraser, E. D, Northup, J. K, Martin, M W., and Harden, T. K. (1987) Identification and purification from bovine brain of a guanine-nucleotide-bindmg protein distinct from Gs, Gi and Go. Biochem. J. 246, 431–439.
Wilson, A. L. and Maltese, W. A. (1995) Coupled translation/prenylation of Rab proteins in vitro, in Methods in Enzymology, vol 250 (Casey, P. J and Buss, J E., eds.), Academic, San Diego, pp. 79–91
Wright, L S. and Siegel, F. L. (1993) Protein methylation in cerebellar synaptosomes J Neurochem 60, 1475–1482
Yamaguchi, T., Shirataki, H., Kishida, S, Miyazaki, M, Mshikawa, J, Wada, K., Numata, S, Kaibuchi, K. and Takai, Y (1993) Two functionally different domains of rabphilin-3A, Rab3A p25/smg p25A-binding and phospholipid and Ca2+-binding domains. J. Biol Chem. 268, 27,164–27,170.
Yeramian, P, Chardin, P, Madaule, P., and Tavitian, A (1987) Nucleotide sequence of human rho cDNA clone 12. Nucleic Acids Res. 15, 1869.
Zahraoui, A., Touchot, N., Chardin, P. and Tavitian, A. (1989) The human Rab genes encode a family of GTP-binding proteins related to yeast YPT1 and SEC4 products involved in secretion. J. Biol Chem. 264, 12,394–12,401
Zerial, M. and Stenmark, H. (1993) Rab GTPases in vesicular transport. Curr. Opin Cell Biol. 5, 613–620.
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Pal Bhullar, R. (1997). Small-Molecular-Weight G Proteins. In: Mishra, R.K., Baker, G.B., Boulton, A.A. (eds) G Protein Methods and Protocols. Neuromethods, vol 31. Humana Press. https://doi.org/10.1385/0-89603-490-9:29
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