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The c-fms proto-oncogene encodes a transmembrane glycoprotein that is probably identical to the receptor for the macrophage colony stimulating factor, CSF-1. Forty C-terminal amino acids of the normal receptor are replaced by 11 unrelated residues in the feline v-fms oncogene product, deleting a C-terminal tyrosine residue (Tyr969) whose phosphorylation might negatively regulate the receptor kinase activity. We show that the human c-fms gene stimulates growth of mouse NIH 3T3 cells in agar in response to human recombinant CSF-1, indicating that receptor transduction is sufficient to induce a CSF-1 responsive phenotype. Although cells transfected with c-fms genes containing either Tyr969 or Phe969 were not transformed, cotransfection of these genes with CSF-1 complementary DNA induced transformation, with c-fms(Phe969) showing significantly more activity than c-fms(Tyr969). In the absence of CSF-1, chimaeric v-fms/c-fms genes encoding the wild-type c-fms C terminus were poorly transforming, whereas chimaeras bearing Phe969 were as transforming as v-fms. Thus, the Phe969 mutation, although not in itself sufficient to induce transformation, activates the oncogenic potential of c-fms in association with an endogenous ligand or in conjunction with mutations elsewhere in the c-fms gene that confer ligand-independent signals for growth.

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NIH 3T3 cells cotransfected with the human c-fms proto-oncogene together with a 1.6-kilobase cDNA clone encoding a 256-amino-acid precursor of the human mononuclear phagocyte colony-stimulating factor CSF-1 (M-CSF) undergo transformation by an autocrine mechanism. The number of CSF-1 receptors on the surface of transformed cells was regulated by ligand-induced receptor degradation and was inversely proportional to the quantity of CSF-1 produced. A tyrosine-to-phenylalanine mutation at position %9 near the receptor carboxyl terminus potentiated its transforming efficiency in cells cotransfected by the CSF-1 gene but did not affect receptor downmodulation. CSF-1 was synthesized as an integral transmembrane glycoprotein that was rapidly dimerized through disulfide bonds. The homodimer was externalized at the cell surface, where it underwent proteolysis to yield the soluble growth factor. Trypsin treatment of viable cells cleaved the plasma membrane form of CSF-1 to molecules of a size indistinguishable from that of the extracellular growth factor, suggesting that trypsinlike proteases regulate the rate of CSF-1 release from transformed cells. The data raise the possibility that this form of membrane-bound CSF-1 might stimulate receptors on adjacent cells through direct cell-cell interactions.

The viral oncogene v-fms encodes a transforming glycoprotein with in vitro tyrosine-specific protein kinase activity. Although most v-fms-coded molecules remain internally sequestered in transformed cells, a minor population of molecules is transported to the cell surface. An engineered deletion mutant lacking 348 base pairs of the 3.0-kilobase-pair v-fms gene encoded a polypeptide that was 15 kilodaltons smaller than the wild-type vfms gene product. The in-frame deletion of 116 amino acids was adjacent to the transmembrane anchor peptide located near the middle of the predicted protein sequence and 432 amino acids from the carboxyl terminus. The mutant polypeptide acquired N-linked oligosaccharide chains, was proteolyticaHly processed in a manner similar to the wild-type glycoprotein, and exhibited an associated tyrosine-specific protein kinase activity in vitro. However, the N-linked oligosaccharides of the mutant glycoprotein were not processed to complex carbohydrate chains, and the glycoprotein was not detected at the cell surface. Cells expressing high levels of the mutant glycoprotein did not undergo morphological transformation and did not form colonies in semisolid medium. The transforming activity of the v-fms gene product therefore appears to be mediated through target molecules on the plasma membrane.Retroviral oncogenes, acquired by recombination between viruses and protooncogenes found in the DNA of normal cells, encode proteins which morphologically transform cultured cells and induce tumors in animals. Several of the known viral oncogene products appear to affect cell growth by mechanisms which mimic the response of cell surface receptors to regulatory polypeptide hormones (or growth factors). For example, the product of the v-sis oncogene is homologous and possibly identical to the A chain of plateletderived growth factor (8, 37) so that cells acquiring v-sis elaborate an autoendocrine signal. In contrast, the v-erbB gene encodes a glycoprotein closely related to a portion of the epidermal growth factor receptor (9) Cotransfection experiments were performed with a plasmid (pSV2) containing the dominant selectable marker, eco gpt; transfected colonies were -selected in hypoxanthineaminopterin-thymidine medium containing mycophenolic acid (24) before metabolic labeling or cell sorting experiments. To allow the spread of SM-FeSV genomes in transfected cultures, cotransfection was also performed with pSM-FeSV DNA and proviral DNA from a molecularly cloned, amphotropic murine leukemia virus (pA-MuLV). Transfected culture supernatants, shown to produce infectious virus with a test for virion-associated, RNA-dependent DNA polymerase, were filtered and used to infect rat NRK cells. Virus infection, polymerase assays, and titration of focus-forming SM-FeSV were performed as described previously (32) with 10-fold serial dilutions of virus-containing culture supernatants.Metabolic radiolabeling of glycoproteins, preparation of cell lysates, and immunoprecipitatation. Metabolic radiolabeling medium consist...