Identification of the ligand-binding regions in the macrophage colony-stimulating factor receptor extracellular domain.
The c-fms gene encodes the receptor for the macrophage colony-stimulating factor (M-CSF), and its extracellular domain consists of five immunoglobulin-like subdomains. To identify which of the five immunoglobulin-like regions are involved in ligand binding, we polymerase chain reaction-cloned five segments of the extracellular domain of the murine c-fms gene, each starting with the normal initiation codon and containing successive additions of the immunoglobulin-like subdomains. These protein segments are designated A, B, C, D, and E and contain, from the N-terminal end, either one, two, three, four, or all five immunoglobulin-like subdomains, respectively. Each segment was expressed as a secreted soluble protein from a baculovirus expression vector in Sf9 insect cells. In addition, segments A, B, C, and E were produced as soluble alkaline phosphatase fusion proteins, as was a segment containing only the fourth and fifth immunoglobulin domains. These segments of the Fms extracellular domain were used to assess M-CSF binding by competition radioimmunoassays, plate binding immunoassays, and immunoprecipitation analyses. The results indicated that the first two N-terminal immunoglobulin-like domains did not interact with M-CSF but, in combination with the third immunoglobulin-like domain, provided high-affinity M-CSF binding. The fourth and fifth immunoglobulin-like domains near the cell membrane did not exhibit M-CSF binding and may inhibit interaction of M-CSF with the first three immunoglobulin domains. These results suggest that the three N-terminal immunoglobulin-like domains constitute the high-affinity M-CSF binding region and that the fourth and fifth immunoglobulin-like domains may perform functions other than ligand binding.
Wang
Z E
ZE
Cell Biology Department, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Myles
G M
GM
Brandt
C S
CS
Lioubin
M N
MN
Rohrschneider
L
L
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004252:DNA Mutational Analysis; D005110:Extracellular Space / Q000378:metabolism; D006031:Glycosylation; D066298:In Vitro Techniques; D008024:Ligands; D016173:Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009036:Moths; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000378:metabolism / Q000648:ultrastructure*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D012995:Solubility; D013329:Structure-Activity Relationship
D008024:Ligands; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D016173:Macrophage Colony-Stimulating Factor; D016186:Receptor, Macrophage Colony-Stimulating Factor