The soluble granulocyte-macrophage colony-stimulating factor receptor's carboxyl-terminal domain mediates retention of the soluble receptor on the cell surface through interaction with the granulocyte-macrophage colony-stimulating factor receptor β-subunit

The hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates its activity through binding to cell-surface receptors. The high-affinity GM-CSF receptor (GMR) consists of two transmembrane-anchored subunits: a ligand-specific, low-affinity subunit (GMR alpha); and a signal-transducing beta-subunit (GMR beta). The human GMR alpha subunit also exists in a soluble isoform (SOL alpha) which antagonizes GM-CSF activity in vitro. Previous studies by us have shown that coexpression of SOL alpha and a mutated GMR beta in BHK cells results in retention of SOL alpha on the cell surface and the formation of an intermediate affinity binding complex (K-d approximately 300 pM). This paper investigates the mechanism of the retention of SOL alpha on the cell surface. The data demonstrate that SOL alpha is anchored by a direct, ligand-independent interaction with GMR beta which also occurs when SOL alpha is coexpressed with wild-type GMR beta. However, SOL alpha and wild-type GMR beta form a complex which binds GM-CSF with high affinity (K-d = 39 pM), indistinguishable from the binding characteristics of the TM alpha/GMR beta complex. The experiments further reveal that the interaction between SOL alpha and GMR beta is abrogated by removal of the unique 16 amino acid carboxyl-terminal domain of SOL alpha. Specific mutation of cysteine 323 in this carboxyl-domain to alanine also eliminates the cell-surface retention of SOL alpha identifying this residue as being necessary for the formation of the SOL alpha/GMR beta complex.