title: Conserved amino acids in the human granulocyte-macrophage colony-stimulating factor receptor-binding subunit essential for tyrosine phosphorylation and proliferation.
abstract: A superfamily of growth factor and cytokine receptors has recently been identified, which is characterized by four spatially conserved cysteine residues and a tryptophan-serine motif (WSXWS) in the extracellular domain and proline-rich cytoplasmic domain. The high-affinity human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, hGM-CSFR, consists of two subunits, alpha (hGM-CSFR alpha), which is required for ligand binding, and beta (hGM-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 mutations in the conserved amino acids of the alpha subunit to determine their function in signal transduction, as assayed by tyrosine phosphorylation and proliferation. Disruption of either of the conserved disulfide bonds in the extracellular domain abolishes low-affinity binding but not binding to a preformed heterodimeric complex with the beta-chain. Cells expressing receptors with mutations in cysteines 2 or 3 grew as well as cells expressing wild-type receptors in human GM-CSF (hGM-CSF) 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. The WSXWS motif and the cytoplasmic domain are required for function of the human GM-CSF receptor, as stable cell lines expressing receptors with these mutations were unable to proliferate continuously in hGM-CSF. Surprisingly, no function for the conserved proline-rich region of the cytoplasmic domain could be ascertained from these studies; cells expressing these receptors were indistinguishable from wild-type in both binding and functional assays.
authors.affiliation: Department of Biologic Chemistry, UCLA School of Medicine 90024.
authors.forename: L V
authors.initials: LV
authors.lastname: Ronco
authors.forename: S E
authors.initials: SE
authors.lastname: Doyle
authors.forename: M
authors.initials: M
authors.lastname: Raines
authors.forename: L S
authors.initials: LS
authors.lastname: Park
authors.forename: J C
authors.initials: JC
authors.lastname: Gasson
mesh_terms: D000595:Amino Acid Sequence; D002455:Cell Division / Q000502:physiology*; D002460:Cell Line; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000032:analysis; D011485:Protein Binding / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction
chemical_list: D010750:Phosphoproteins; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases