Ligand-independent Cell Surface Expression of the Human Soluble Granulocyte-Macrophage Colony-stimulating Factor Receptor α Subunit Depends on Co-expression of the Membrane-associated Receptor β Subunit

Abstract

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.

Interleukin-5 Receptor Subunit Oligomerization and Rearrangement Revealed by Fluorescence Resonance Energy Transfer Imaging

“…The ability of the IL-5R␣ subunit to bind ligand even in the absence of ␤c, albeit at a lower affinity (9), supports the notion that ligand-linked conformational changes occur in one or more chains of the heteromeric receptor-ligand complex. Since ␤c is the shared signaling receptor subunit for IL-3, IL-5, and GM-CSF, the rearrangement of preformed ␤c subunits could be a common feature in the activation of IL-5, IL-3, and GM-CSF receptor systems, although there may be differences in the assembly processes among the three ␤c cytokines, as hinted by the observation of preassembled GM-CSFR␣-␤c (66,67). We believe that the cell FRET approach developed here offers the potential to compare cell surface receptor assembly and activation processes among the ␤c cytokines.…”

Soluble receptors in human disease

“…A fifth mechanism of soluble receptor action has been suggested by the recent finding that the soluble GM-CSF ␣ receptor can associate with its ␤ subunit only when the two subunits are synthesized in the same cell (Fig. 1E) [117]. The soluble ␣ subunit and the ␤ subunit are unable to associate when soluble receptor is added exogenously, suggesting that the association is a result of intracellular protein processing.…”

Antibody Cross-linking of the Glycosylphosphatidylinositol-linked Protein CD59 on Hematopoietic Cells Induces Signaling Pathways Resembling Activation by Complement

“…Flow Cytometry-Cells were prepared for flow cytometry as described previously (30). Primary antibody incubations were done with 0.5 g/ml anti-CD59 antibody (rat anti-human CD59 YTH53.1 or mouse anti-human CD59 MEM43; Serotec, Oxford, UK).…”