Hematopoietic growth factor (HGF) receptors are the first link in the relay of signals that culminate in proliferation or differentiation of a progenitor hematopoietic cell. Recent advances in signal transduction suggest that the majority of receptors interact with overlapping sets of secondary effectors inside the cell. It is therefore remarkable that seemingly opposite responses -proliferation and differentiation- can be signaled through shared effector proteins, and that specificity in response to a given HGF can be maintained. How this is achieved is unknown. Two model systems will be developed to address these questions. In the first model, the rat pheochromocytoma cell line PC12 will be used to study the function of the mouse interleukin-3 receptor (IL-3R). Preliminary data suggest that introduction of the IL-3R components into PC12 cells, and stimulation with IL-3 leads to neuronal differentiation. Thus, structure-function relationship of the IL-3R and the role of other HGF and HGF chimeric receptors can be assessed in the absence of interfering endogenous receptors, and in the context of differentiation rather than proliferation. Chimeric receptors have been used to define critical regions in HCF receptors mediating specificity in the differentiation response. For instance, an EPO-R/betaIL-3R chimeric receptor can induce limited differentiation of a progenitor cell line, even though it lacks the cytoplasmic region of the EPO-R. One potential caveat in these experiments is the fact that the indicator cell line is only permissive to limited differentiation, and may already be precommitted to a given lineage. Thus, the observed differentiation may not reflect a physiological response. To circumvent this limitation and address the question of specificity, embryonic stem (ES) cell clones will be obtained in which the wild-type erythropoietin (EPO) receptor has been replaced by an EPO-R/betaIL-3R chimeric receptor. The contribution of this chimeric receptor to blood formation and more specifically to erythropoiesis will be monitored in vivo in mice homozygous for the chimeric receptor, and, in vitro, in colony assay with progenitor cells derived from embryoid bodies. These two model systems described above represent a novel approach to assay HGF-R function, and complement or improve existing models that rely primarily on proliferation.
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