This revised application for five years of continuing funding is to designed to study the role of the Gm-CSF receptor in normal and in malignant hemopoiesis. The research plan consists of two specific aims. In the first specific aim, Dr. Golde plans to study the function of the GM-CSF receptor. Based on a large number of previous studies and preliminary data, the first specific aim will investigate the function of the membrane-bound form of the GM-CSF alpha subunit in glucose transportation. Previous studies have shown that in contrast to proliferation, glucose uptake can be signaled in response to GM-CSF through the low affinity GM-CSF receptor alpha subunit alone. The mechanism of this effect will be studied by altering the alpha subunit density on both Xenopous and human cell lines (leukemic and melanoma), perturbations of he more classical alpha/beta heterodimeric receptor signaling complex using metabolic inhibitors and antisense oligonucleotide strategies, and the effect of he soluble GM-CSF receptor alpha subunit. These studies will then be extended to determine whether the IL-5 and Il-3 alpha subunits function in a similar manor in glucose transport. The GM-CSF receptor alpha subunit will then be mutated in order to determine the functional domains responsible for interacting with glucose transporters and signaling increased glucose uptake in response to GM-CSF. Additional studies will be conducted to determine the function of the alpha-beta complex in oocytes, COS cells and HL60 models. In the Xenopous studies, an attempt will be made to directly ascertain the components of the signaling complex. Specifically, reconstitution experiments using the alpha and beta chains of the GM-CSF receptor will be generated. If signaling through tyrosine and serine/threonine kinases does not ensue, a cloning strategy will be undertaken to determine the missing signaling components. In this way, it is hoped that the entire signaling cascade will be dissected. A direct role for putative intracellular signaling molecules will then be undertaken using an antisense oligonucleotide knock out strategy in both the Xenopous and leukemic cell systems. Function of the soluble GM-CSF receptor alpha subunit will next be determined by studying the relative distribution of the soluble and membrane-bound forms of the polypeptide, the effect of soluble GM-CSF receptor alpha on GM-CSF-mediated signal transduction, the inverse effect of GM-CSF on expression of the soluble form of its receptor and structure-function studies of the soluble form to determine its role in the previously studied properties. In the second specific aim, the role of GM-CSF in signaling through to nuclear events will be studied. Early gene activation in the form of EGR-1/TIS8 will be assessed in response to GM-CSF activation of cells by study of promoter activity of this early response gene.
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