Hematopoietic growth factors stimulate cell growth by interacting with specific receptors. Stem Cell Factor (SCF) is a novel growth factor that acts on hematopoietic stem cells and progenitor cells. The receptor for SCF is encoded by the c-kit proto-oncogene. We propose to characterize the c-kit receptor on hematopoietic and non-hematopoietic cells. The broad, long-term objectives of this proposal are to provide a better understanding of the mechanism of action of SCF in normal and neoplastic hematopoiesis and to provide information about the structure and function of the c-kit receptor.
The specific aims are as follows. First, we will determine the binding properties, abundance, cellular distribution and size of the c-kit protein on both normal hematopoietic cells, marrow microenvironmental cells, and neoplastic cells. Three approaches will be used to achieve this aim: (a) equilibrium binding studies with 125I-SCF to quantitate receptor numbers and binding affinity on populations of cells plus autoradiography to permit analysis of 125I-SCF binding to individual cells, (b) isolation and characterization of populations of receptor-bearing cells using our anti-c-kit receptor antibody SR-1, and (c) affinity crosslinking and ligand blotting to determine the size and subunit structure of the receptor. We will compare the structure of the c-kit receptor on normal and neoplastic cells. Second, we will generate additional anti-c-kit receptor monoclonal antibodies by immunizing mice with cells expressing high numbers of c-kit receptors or with purified soluble c-kit protein. Our hybridoma screening techniques are designed to identify both neutralizing and nonneutralizing antibodies. We will use the antibodies to map functional domains of the c- kit receptor. Third, we will identify the ligand binding domain of the c-kit receptor and the regions required for receptor dimerization using interspecies human- mouse hybrid c-kit receptors and truncated receptors. The ability of these mutant receptors to bind human and rat 125I-SCF, to dimerize, to autophosphorylate, and to transmit a proliferative signal to the cell will be determined.
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