Characterization of the soluble human granulocyte-macrophage colony-stimulating factor receptor complex

J. F. DiPersio, C. Hedvat, C. F. Ford, D. W. Golde, J. C. Gasson

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GM-R) is expressed on both hematopoietic and non-hematopoietic tissues. Although the receptor has been identified by crosslinking studies as an 84,000-dalton protein, very little is known about its biochemistry. In this report, we describe a soluble binding assay for the human GM-R which allowed us to characterize the receptor complex from various sources, including plasma membranes of placenta, neutrophils, and human myeloid leukemia cell lines. Preparation of membranes as well as solubilization by Triton X-100 and N-octylglucoside resulted in a 5- 10-fold lower affinity of the receptor for GM-CSF. The K(d) decreased from 20 to 80 pM in intact cells to 200-500 pM in both intact and solubilized membranes. Binding in solution was rapid, specific for GM-CSF, and best fit a 'one-site' model with an approximate K(d) of 500 pM. The dissociation rate constant for the soluble GM-R was very similar to that of intact cells (k2 = 0.013 min-1 versus 0.017 min-1 respectively). As expected, solubilized membranes obtained from those cells expressing the highest number of GM-R (neutrophils and dimethyl sulfoxide-induced HL-60 cells; ~ 500-800 sites/cell) possessed the highest concentration of soluble GM-R (~ 2-3 x 108 GM-R/μg). Cross-linking of 125I GM-CSF to soluble GM-R resulted in the appearance of two specifically labeled complexes. A major 110-kDa receptor-ligand complex is found when cross-linking is performed with intact cells; both 110- and 200-kDa species are seen when cross-linking is performed with either intact membranes or soluble GM-R. These studies define methods by which intact GM-R can be solubilized and measured in solution, permitting a more complete biochemical characterization of the intact GM-R complex.

Original languageEnglish
Pages (from-to)279-286
Number of pages8
JournalJournal of Biological Chemistry
Volume266
Issue number1
StatePublished - Jan 31 1991
Externally publishedYes

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