Characterization of the soluble human granulocyte-macrophage colony-stimulating factor receptor complex.
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 cross-linking 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 Kd 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 Kd 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; approximately 500-800 sites/cell) possessed the highest concentration of soluble GM-R (approximately 2-3 x 10(8) GM-R/micrograms). 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.
DiPersio
J F
JF
Department of Medicine, UCLA School of Medicine 90024-1678.
Hedvat
C
C
Ford
C F
CF
Golde
D W
DW
Gasson
J C
JC
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents; D005260:Female; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007938:Leukemia; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D003902:Detergents; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor