I wish to test the hypothesis that individual signal transduction systems are mediated by unique plasma membrane GDP-binding proteins. Previous reports, using anti-ras monoclonal Y13-259 have implied this oncogenic protein may be involved in coupling receptor-mediated phosphatidylinositol turnover and the generation of a proliferative responses to either oncogenic tyrosine kinase transformation or normal cellular response to serum addition. Coupling of cell surface receptors to the generation of intracellular second messengers theoretically occurs through the accelerated exchange of bound GDP for GTP by receptor specific G-proteins. Utilizing high resolution anion exchange chromatography, we have discovered at least ten plasma membrane GOP-binding proteins, five of which react with Y13-259. Growth factor dependent changes in the GDP off-rates for each individual GDP-binding species will be examined. Positive coupling between a specific growth factor receptor and a unique GDP-binding protein will be monitored by accelerated rates of quanine nucleotide exchange. Practically, this will be apparent by the disappearance of a single GDP-binding protein in the elution profile from a high resolution anion exchange column. Possible ATP dependent and tyrosine kinase dependent changes in GDP off-rates will be examined. Tyrosine kinase dependent guanine nucleotide exchange will also be examined in membranes containing a temperature sensitive src protein. Requirements for soluble guanine nucleotide exchange factors will also be analyzed. GDP-binding proteins purified from brain tissue will be used in the development of monoclonal antibodies against proven coupling factors. These antibodies will be used in microinjection experiments to determine if single coupling events are sufficient to generate a proliferative signal. Reconstitution experiments will also be done using purified unique plasma membrane G-proteins and GDP-binding protein deficient solubilized membranes. The native molecular weights, possible subunit interactions, pertussis/cholera toxin catalyzed ADP-ribosylation, and similarities to genetically defined ras-related oncogene families will also be evaluated.
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