The ultimate goal of this research project is to determine the genetic and molecular basis of receptor-mediated signal transfer mechanisms of polypeptide hormones and growth factors, particularly insulin and epidermal growth factor (EGF) in tissue cultured cells. We are also interested in the growth promoting activity of the tumor promoters. We are approaching the proposed goals through the use of somatic cell mutagenesis in combination with molecular biological and biochemical techniques. By selecting mutants that are defective or extraordinary in the various steps of the mechanisms involved in the action of insulin, EGF and tumor promoters, we hope to define and characterize the pathway both genetically and biochemically. Furthermore, we utilize these genetic variants to map genes that are involved in hormone's binding, internalization, and intracellular processing, receptor's clustering and recycling, and various post-receptor functions by cell hybridization analysis.
Specific aims of our investigation are the following: (1) continued characterization of the isolated genetic variants to determine their genetic and biochemical lesions; (2) intracellular fate of the hormone-toxin conjugates to better understand their entry mechanisms; (3) isolation of new series of variants by the application of cytotoxic conjugates to the mutagenized cell populations which are synchronized at a certain stage of cell cycle or temporarily treated with specific inhibitors of endocytic mechanisms; (4) formation of cell hybrids between the isolated variants or with other cells to perform complementation group analysis and chromosomal localization of the corresponding genes; (5) mapping of the human receptor genes for insulin, TGFs, MSA, and IGFs by crosslinking techniques; (6) studies on the genetic and molecular basis of the EGF receptor-overproduction in A431 cells, particularly the relation between translocation chromosome M4 and production of truncated variant 3kb mRNA and 110kd EGF receptor fragments; (7) examination of the possible chromosome rearrangements and gene alterations in various human tumor-derived cells which are overproducing or deficient in EGF receptors using cDNA probes for EGF receptor/erbB oncogene. The recent discovery of a family of transforming growth factors in relation to oncogene products implies a complex genetic regulation of their receptor systems. Although the cell genetic approach to these receptor systems is still in its infancy, it provides unique experimental systems by which regulation of cell growth is studied.
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