The c-fins 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-fins 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. The macrophage colony-stimulating factor (M-CSF) has been identified as the key lineage-specific hematopoietic growth factor that acts directly on monocytes/macrophages and their progenitor cells to stimulate their survival, proliferation, differentiation, and mature cell functions (20). M-CSF is a disulfide-linked homodimer synthesized as a transmembrane molecule and proteolytically processed to produce the soluble form (19,30,31). Both membrane-bound and soluble M-CSF can stimulate target cell growth, and the biologically active portion of the ligand is contained in the amino-terminal 150 amino acids of the approximately 550-amino-acid full-length form (16,49). Structural studies of M-CSF have indicated that the disulfide-bonded dimer configuration is essential for activity but that the presence of carbohydrate on the molecule is not needed.The receptor for M-CSF is a member of the tyrosine kinase class of growth factor receptors and is identical to the protein product of the c-fins proto-oncogene (Fms) (40 the c-kit proto-oncogene, several fibroblast growth factortype receptors, the Flk receptors, and the Drosophila torso gene product (5, 41, 47, 52). In addition, Fms is a member of a broader class of receptor molecules defined by the existence of immunoglobulin-like structural subdomains within the extracellular region of the receptor (58). This group includes receptors containing multiple immunoglobulin-like subdomains such as NCAM, ICAM (15), the membranebound form of immunoglobulin M, the CD4 antigen, the interleukin-1 receptor (42), and the a and 1 subunits of the T-cell receptor. Those receptors with a single immunoglobulin-like subdomain such as the a and 1 subunits of the interleukin-6 receptor, interleukin-7 receptor, LIF (leukemia inhibitory factor) receptor, and the granulocyte CSF receptor also can be considered members of this group (2,7,8,11).Signal transduction and other effector functions of Fms initiate with M-CSF binding to a single high-affinity site in the extracellular domain of each of two Fms molecules (reviewed in reference 59). This M-CSF-ind...

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.