The long-term aims of this project are to understand the control of the production and function of white blood cells (particularly granulocytes and macrophages) at both the cellular and molecular level in order to devise improved therapies for the treatment of blood disorders and leukemia. Preliminary results using site-directed mutants of the cytoplasmic domain of the GM-CSF receptor beta-chain have suggested that the multiple actions of GM-CSF have their origins from different cytoplasmic modules within the receptor that initiate separate pathways leading to cell survival, proliferation, differentiation or clonal suppression of proliferation. Sin each of these pathways can be subverted in the generation of leukemias and since one pathway may be utilized by several different cytokines, the most selective therapeutics of the future will target individual cytoplasmic signaling modules. The major aims of the current proposal are to use two cytokine/receptor systems (GM-CSF and LIF) to unequivocally map specific receptor cytoplasmic domains and biochemical pathways to particular biological responses. A panel of receptor truncation, tyrosine and chimeri mutants will be expressed in a series of cell lines in which survival, macrophage differentiation, proliferation and clonal suppression can be measured. This work will be extended to in vivo studies with normal hemopoietic cells by transient transfection or long-term reconstitution of bone marrow cells from mice engineered to lack a functional GM-CSF receptor beta-chain gene and so can be forced to express only the mutated receptors. A molecular genetic approach will be used to identify the functionally necessary biochemical components of the LIF-induced differentiation pathway. LIF unresponsive M1 myeloid leukemic cells will be generated using selectable genetic elements and classified according to whether they are dominant or recessive mutants and to which complementation group they belong by cell fusion. LIF-responsiveness will then be rescued, again using selectable genetic elements, by transfection o infection with cDNA expression libraries and the critical mutated or delete proteins thus identifies.
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