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

Elizabeth W Murray,Carin Pihl,Annette Morcos,Christopher B Brown

doi:10.1074/jbc.271.26.15330

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.

Highlights

Another common feature of many cytokine receptors is the presence of GM-CSF as demonstrated using flow cytom- formation of soluble isoforms
Cell Lines Expressing GM-CSF receptor (GMR)␤—To examine the interaction between exogenously added solGMR␣ and GMR␤ in the presence of GM-CSF, we developed a BHK cell line that stably expresses GMR␤
We have shown that exogenous solGMR␣ in the presence of GM-CSF is incapable of interacting with GMR␤ expressed on the cell surface

Summary

Introduction

Another common feature of many cytokine receptors is the presence of GM-CSF as demonstrated using flow cytom- formation of soluble isoforms (reviewed in Ref. 10). Because the GMR␤ (A 3 C) clone is capable of forming functional high affinity receptors with transmembrane GMR␣ (Kd ‫ ؍‬64 pM), we feel that the cell surface retention of solGMR␣ is independent of the GMR␤ mutation. The generation of soluble isoforms is common for many cytokine receptors, the mechanism used to generate them varies They include alternative splicing of RNA transcripts, proteolytic cleavage of extracellular domains, and cleavage of glycosyl-phosphatidyl-inositol membrane anchors. Current evidence suggests that the ␤ subunit can transduce signals; controversy remains over the GMR␣ subunit’s role in signaling [17,18,19,20,21,22]

Methods

Results

Conclusion