Ligand-independent cell surface expression of the human soluble granulocyte-macrophage colony-stimulating factor receptor alpha subunit depends on co-expression of the membrane-associated receptor beta subunit.
The hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates its activity through binding to cell surface receptors. The receptor for GM-CSF belongs to a superfamily of cytokine receptors characterized by a conserved extracellular motif. The high affinity GM-CSF receptor (GMR) consists of two transmembrane anchored subunits; a ligand binding alpha subunit (transmembrane GMRalpha) and a signal transducing beta subunit (GMRbeta), both of which belong to the cytokine receptor superfamily. The human GM-CSF receptor alpha subunit also exists in a soluble form (solGMRalpha), which antagonizes GM-CSF activity in vitro. We directly tested the potential for solGMRalpha to interact with GMRbeta in vitro. Our experiments demonstrated that exogenous solGMRalpha, even in the presence of GM-CSF, does not interact with GMRbeta on the cell surface. However, when solGMRalpha and GMRbeta are co-expressed in baby hamster kidney cells, solGMRalpha is retained on the cell surface and forms a functional intermediate affinity GM-CSF binding complex (Kd = 331 pM). In addition, the cell surface expression of solGMRalpha is independent of the presence of GM-CSF as demonstrated using flow cytometry. Cells expressing only solGMRalpha do not show cell surface retention or form functional GM-CSF cell surface binding complexes. Sequencing of our GMRbeta clone revealed a nucleotide substitution (A --> C) resulting in the substitution of Ala for Glu at position 9 from the amino terminus of the mature GMRbeta peptide. Because the GMRbeta (A --> C) clone is capable of forming functional high affinity receptors with transmembrane GMRalpha (Kd = 64 pM), we feel that the cell surface retention of solGMRalpha is independent of the GMRbeta mutation. We suggest that the co-expression and interaction of solGMRalpha and GMRbeta represents a previously unrecognized GM-CSF receptor complex and a novel, ligand-independent mechanism of cytokine receptor assembly.
Murray
E W
EW
Department of Medicine, Cancer Biology Research Group, The University of Calgary, Calgary, Alberta, T2N 4N1 Canada.
Pihl
C
C
Morcos
A
A
Brown
C B
CB
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D008024:Ligands; D046911:Macromolecular Substances; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins; D012995:Solubility; D014162:Transfection
D008024:Ligands; D046911:Macromolecular Substances; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor