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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK044194-02
Application #
3245710
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1992-09-30
Project End
1997-09-29
Budget Start
1993-09-30
Budget End
1994-09-29
Support Year
2
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Washington
Department
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Sabath, D F; Lin, N; Sabath, D E et al. (2000) Tyrosine 462 of the membrane-proximal F'-G' loop of murine Mpl is not essential for high-affinity binding of thrombopoietin. Cytokine 12:127-33
Broudy, V C; Lin, N L; Liles, W C et al. (1999) Signaling via Src family kinases is required for normal internalization of the receptor c-Kit. Blood 94:1979-86
Broudy, V C; Lin, N L; Buhring, H J et al. (1998) Analysis of c-kit receptor dimerization by fluorescence resonance energy transfer. Blood 91:898-906
Broudy, V C; Lin, N L; Sabath, D F et al. (1997) Human platelets display high-affinity receptors for thrombopoietin. Blood 89:1896-904
Broudy, V C (1997) Stem cell factor and hematopoiesis. Blood 90:1345-64
Broudy, V C; Lin, N L; Fox, N et al. (1996) Thrombopoietin stimulates colony-forming unit-megakaryocyte proliferation and megakaryocyte maturation independently of cytokines that signal through the gp130 receptor subunit. Blood 88:2026-32
Broudy, V C; Lin, N L; Priestley, G V et al. (1996) Interaction of stem cell factor and its receptor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spleen. Blood 88:75-81
Kaushansky, K; Lin, N; Grossmann, A et al. (1996) Thrombopoietin expands erythroid, granulocyte-macrophage, and megakaryocytic progenitor cells in normal and myelosuppressed mice. Exp Hematol 24:265-9
Turner, A M; Lin, N L; Issarachai, S et al. (1996) FLT3 receptor expression on the surface of normal and malignant human hematopoietic cells. Blood 88:3383-90
Broudy, V C; Lin, N L; Kaushansky, K (1995) Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro. Blood 85:1719-26

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