Cell growth, cell surface receptor signaling, signal transduction mechanisms, recognition domains and drug design.
Ras and heterotrimeric GTP binding proteins are major components of cell growth signaling pathways that are triggered by ligand-activated tyrosine kinase or seven-transmembrane-domain receptors. Both Ras proteins and the g subunits of heterotrimeric G pro teins contain a carboxyl-terminal S-prenylcysteine (SPC) which is absolutely required for their membrane anchorage and biological activities as mediators in signal transduction. Independent studies performed by us and by others revealed that prenylcystein e mimetics specifically interfere with the interactions of activated seven transmembrane-domain receptors and heterotrimeric G proteins as well as with the membrane anchorage and functions of Ras proteins.
One of our major goals is the identification of a presumably distinctive Ras SPC-docking site and the characterization of its role and significance in Ras-mediated cell-growth signaling. We have recently demonstrated for the first time the selective dislo dging of Ras from intact Ras-transformed cell membranes by the unique SPC mimetic, S-farnesylthiosalysilic acid (FTS). Consequently, the cell’s transformed-phenotype is reversed, and cell- and tumor-growth in nude mice are inhibited. Structure activity re lationships among FTS analogues, and selective inhibition of growth signals upstream, but not downstream, of Ras demonstrate FTS specificity and selectivity. These results provide a strong support for the existence of distinctive, yet undetected, SPC anch orage sites for Ras in cell membranes, as well as new experimental tools for the identification of such sites, and a novel concept of competing Ras from its sites as a potential method for cancer chemotherapy. We attempt now to demonstrate that Ras or het erologous proteins carrying the Ras docking sequence can be dislodged from the plasma membrane by specific SPC mimetics even without an effect on Ras processing, to determine the consequences of this effect on the stability of the dislodged Ras and on the activity of down-stream growth signaling components and on cell growth and attempt to label, tag, identify and isolate the Ras docking site. FTS and other SPC mimetics interfering with Ras functions, are used to affect Ras-mediated activation of c-Raf1 k inase and mitogen-activated protein kinase (MAPK), in Rat1 fibroblasts stably expressing activated Raf, Ras, tyrosine kinase receptors, partially processed Ras and Raf targeted to the membrane by a Ras targeting signal sequence (Raf-CTR). The nature of th e FTS-elicited dislodging of various ras mutants and of Raf-CTR from cell membranes, and the fate of the dislodged proteins are studied by combined immunoprecipitation/immunoblot assays. The relationships between Ras-dislodging and the structure of SPC-m imetics and their effects on Raf-MAPK is now being determined. These experiments, together with radioligand binding assays ([3H]FTS) and affinity labeling ([3H]FTS-mustard) combined with electrophoretic and chromatographic methods should lead to identific ation of SPC sites for Ras.
A second major goal in our studies is the identification of SPC sites for Gbg. We perform functional analysis of the effects of SPC mimetics on the interaction of G protein bg subunits with known regulatory proteins (e.g. guanine nucleotide exchange facto rs, GTPase-regulating proteins, downstream targets like components of the pathway for Ras proteins). In these studies we use both intact cells, that express Gi/Go proteins and seven-transmembrane-domains-receptors (muscarinic, LPA, thrombine, receptors), and cell free systems where G proteins, phospholipase-C(, hSosl and K-RasB purified from baculovirus-infected insect cells are reconstituted. Our studies are expected to provide new information on the nature and on the structural properties of SPC domains and could help in the design of new strategies to alleviate certain types of human cancers and to block undesired cell proliferation in many other types of human diseases.