Colony-stimulating factors (CSFs) are required for in vitro growth of myeloid colonies in semi-solid systems. There is considerable clinical evidence to suggest that these glycoprotein mediators are the physiological regulators of human granulopoiesis. Little is known about the molecular characteristics and mechanism of action of human CSFs because purified protein has not been available. Recently we have purified human granulocyte-macrophage CSF (GM-CSF) and obtained cDNA clones which are efficiently expressed in COS monkey cells. With the availability of these reagents it is now possible to investigate the physiological functions of this important hematopoietic regulator. Based on the fact that GM-CSF is active in the picomolar range, it is likely to exert its effects through binding to a specific receptor. Recombinant (biosynthetic) GM-CSF will be used to study specific binding to responsive target cells that have been identified in biological assays. The specific binding assay will also be used to identify the target cells in normal human bone marrow and peripheral blood, as well as the presence of high affinity binding sites on cells isolated from patients with various myeloproliferative disorders. The molecular weight and subunit structure of the GM-CSF receptor will be characterized by affinity labeling. The receptor will be purified from a human cell line which is found in the specific binding assay to have a high number of receptors. Membranes will be prepared and the proteins solubilized and fractionated on a GM-CSF-affinity column. Proteins bound to the column will be eluted with salt and further fractionated by gel filtration. Purification will be monitored by binding of ?125?I-GM-CSF and SDS polyacrylamide gel electrophoresis. Amino acid sequence from the N-terminus of the purified receptor will be used to construct oligonucleotide probes. A cDNA library prepared from mRNA enriched for sequences contained in cells which express the GM-CSF receptor will be screened with the oligonucleotide probes. Positive clones will be verified by Northern analysis and DNA sequencing. Studies will be initiated to examine the events following binding of GM-CSF to the receptor such as receptor internalization, kinase activities, and possible second messengers. Ultimately, genetic variants resistant to GM-CSF action will be used to establish the role of specific pathways in GM-CSF action. (J)
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