Human interleukin-3 (IL-3) is a pluripotent colony stimulating factor (CSF) which has a crucial role in the development of early hematopoietic progenitor cells and a role in some forms of acute lymphocytic leukemia. In order to understand the mechanism of action of IL-3 in normal human hematopoiesis as well as in leukemogenesis it is important to obtain a full biochemical and molecular analysis of the IL-3 receptor and its associated transducing mechanism in normal hematopoietic cells. This application proposes to obtain such a characterization including the cloning of a cDNA(s) for this receptor in a normal human hematopoietic cell. Although the receptor appears in low abundance on human monocytes, the recent availability of expression cloning techniques has made it possible to clone such low abundance proteins. A cDNA library has been constructed from human monocytes in a high efficiency animal cell expression vector (pcDNA1) and pools of these clones will be transfected into COS-1 cells. The transfected cells will be screened for [125I] IL-3 binding. After identifying the cDNA clone coding for the IL-3 receptor the DNA sequence of the clone will be determined and compared to sequences of known genes to determine homologies. The affinity constant of the receptor transfected into COS cells will be compared to that of the natural receptor. The application also proposes to use polymerase chain reaction cDNA amplification as a supplement to expression cloning. If the expression cloning is successful this technique can also be used to identify unique unidentified but related family members to the IL-3 receptor in human monocytes. Amino acid sequence for the IL-3 receptor and the GM-CSF receptor beta subunit deduced from the cDNA sequence, will be used to synthesize peptides for production of monospecific polyclonal antibodies in rabbits. The ability of these antibodies to precipitate the natural and recombinant proteins will be assessed using [35S] methonine labelled cells. When we have an antibody that is successful in precipitating the [35S] methonine labelled IL-3 receptor we will also attempt to precipitate the receptor bound to IL-3. This experiment should reveal whether or not the binding of IL-3 to its receptor induces the association of any accessory proteins with the IL-3 receptor. Finally, based on preliminary data indicating a role for protein kinase C in IL-3 signal transduction, the application proposes to examine the relationship of protein kinase C to IL- 3 mediated signal transduction. Examination of protein phosphorylation and phospholipid hydrolysis in response to the stimulation of human monocytes by IL-3 will be conducted. Taken together these experiments should dramatically increase our understanding of the mechanism of action of human IL-3, and should suggest further experiments to identify molecular mechanisms fundamental to the regulation of normal human hematopoiesis and leukemogenesis.
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