Regulated entry into and withdrawal from the cell cycle occurs in response to a variety of endogenous and exogenous mitogenic signals. Proper cell cycle control is essential to embryogenesis, organotypic growth, differentiation and regeneration. Aberrant mitogenic responses are causal in many pathologies; e.g., developmental abnormalities and neoplasias. Mitogens stimulate many cellular physiological and biochemical changes, collectively termed the """"""""pleiotypic response"""""""". What are the causal steps, within the mitogenic pleiotypic response, in the transition from a resting to a proliferating cell? Do different mitogens (i) activate the same, ubiquitous sequence of events, or do they (ii) activate unique, but converging pathways? To investigate these questions, components of the pleiotypic response have been characterized in variant 3T3 lines which no longer respond mitogenically to either the polypeptide mitogen EGF or the macrocyclic tumor promoter TPA. To expand these studies, additional variants unable to respond to combined administration of EGF and TPA will be isolated by colchicine selection. Variants in the EGF receptor system will be isolated with EGF-toxin conjugates, and characterized for their biochemical defects. TPA modulates cell division, differentiation, and transformation. Intersecting molecualr mechanisms for these phenomena are suggested by (i) homology between growth factor genes and oncogenes, (ii) tyrosine kinase activity of oncogene products and growth factor receptors, and (iii) a common phosphorylation substrate in virally transformed, mitogen-stimulated and TPA-responsive cells. The causal roles of TPA responsive genes will be examined by (i) cloning of cDNAs for genes transcriptionally responsive to TPA in 3T3 cells, (ii) isolating the genes from genomic libraries, and (iii) transfection into TPA nonresponsive varients. The gene products will be characterized with antisera to proteins produced by expression vectors. TPA activates c-kinase, a calcium phospholipid-dependent protein kinase. C-kinase is also activated (via diacylglycerol) by many regulatory agents, including EGF. The c-kinase gene will be cloned, as a first step in defining its causal roles. C-kinase can (perhaps) be considered the """"""""TPA receptor"""""""". Isolation of genes for growth factor receptors will be of great value in elucidating their modes of action. This application also proposes a generalized protocol to clone genes for membrane receptors and a critical test of the protocol; cloning of the EGF receptor gene.
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