The objectives of our proposal are twofold: a) to investigate the mechanism of c-kit receptor activation/function and b) to determine the molecular basis of the pleiotropic functions of c-kit, i.e. to elucidate the mechanism of the control of expression of the c-kit gene. The proto-oncogene c-kit is the normal cellular homolog of the oncogene v- kit of the HZ4 - feline sarcoma virus (Besmer et al., 1986) and encodes a transmembrane tyrosine protein kinase receptor. c-kit has been determined to be allelic with the dominant white spotting locus (W) of the mouse. Mutations at the W locus affect various aspects of hematopoiesis and the proliferation and/or migration of primordial germ cells and melanoblasts during development. The recent analysis of several dominant E mutations revealed that they are dominant negative mutations providing clues about the mechanism of kit mediated signal transduction. We will use an in vitro mutagenesis approach to further define the mechanism of activation of the c-kit tyrosine kinase receptor and of the P80gag-kit transforming protein. A most interesting aspect of the c-kit/W receptor system is its pleiotropy. c-kit function is the result of a combination of factors, namely, availability of ligand, the expression of the c-kit receptor and a functioning downstream signaling pathway. An important concern of our proposal is to further our understanding of the mechanism of the control of c-kit expression at the level of transcription and post transcriptional processing. Tissue specific regulatory sequences in c-kit will be identified by investigating transcription start sites, determining DNaseI hypersensitive sites, determining the transcription rates, by deletion mapping of the 5' flanking region of the c-kit gene with receptor gene constructs and by performing binding experiments to identify cell specific nuclear transacting factors. We will also attempt to create a mutation in a tissue specific promoter element of c-kit in the mouse germ line by use of homologous recombination technology and embryonic stem cells.
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