CONSERVED AMINO-ACIDS IN THE HUMAN GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR RECEPTOR-BINDING SUBUNIT ESSENTIAL FOR TYROSINE PHOSPHORYLATION AND PROLIFERATION

A superfamily of growth factor and cytokine receptors has recently bee n identified, which is characterized by four spatially conserved cyste ine residues and a tryptophan-serine motif (WSXWS) in the extracellula r domain and proline-rich cytoplasmic domain. The high-affinity human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, hG M-CSFR, consists of two subunits, alpha (hCM-CSFR alpha), which is req uired for ligand binding, and beta (hCM-CSFR beta), which is required for signal transduction. Both the alpha and beta subunits are members of the cytokine receptor superfamily. In this study, we analyzed mutat ions in the conserved amino acids of the alpha subunit to determine th eir function in signal transduction, as assayed by tyrosine phosphoryl ation and proliferation. Disruption of either of the conserved disulfi de bonds in the extracellular domain abolishes low-affinity binding bu t not binding to a preformed heterodimeric complex with the beta-chain . Cells expressing receptors with mutations in cysteines 2 or 3 grew a s well as cells expressing wild-type receptors in human CM-CSF (hGM-CS F) and phosphorylated the same proteins on tyrosine residues, although the level of phosphorylation may be attenuated; cysteine 3 appears to be required for generation of the true high-affinity binding site. Th e WSXWS motif and the cytoplasmic domain are required for function of the human GM-CSF receptor, as stable cell lines expressing receptors w ith these mutations were unable to proliferate continuously in hGM-CSF . Surprisingly, no function for the conserved proline-rich region of t he cytoplasmic domain could be ascertained from these studies; cells e xpressing these receptors were indistinguishable from wild-type in bot h binding and functional assays.