Carl W. Rettenmier

The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF 1

The feline c-fms proto-oncogene product is a 170 kd glycoprotein with associated tyrosine kinase activity. This glycoprotein was expressed on mature cat macrophages from peritoneal inflammatory exudates and spleen. Similarly, the receptor for the murine colony-stimulating factor, CSF-1, is restricted to cells of the mononuclear phagocytic lineage and is a 165 kd glycoprotein with an associated tyrosine kinase. Rabbit antisera to a recombinant v-fms-coded polypeptide precipitated the feline c-fms product and specifically cross-reacted with a 165 kd glycoprotein from mouse macrophages. This putative product of the murine c-fms gene exhibited an associated tyrosine kinase activity in immune complexes, specifically bound murine CSF-1, and, in the presence of the growth factor, was phosphorylated on tyrosine in membrane preparations. The murine c-fms proto-oncogene product and the CSF-1 receptor are therefore related, and possibly identical, molecules.

Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and downmodulation of CSF-1 receptors in NIH 3T3 cells transformed by cotransfection of the human CSF-1 and c-fms (CSF-1 receptor) genes.

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 969 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.

Differential processing of colony-stimulating factor 1 precursors encoded by two human cDNAs

The biosynthesis of macrophage colony-stimulating factor 1 (CSF-1) was examined in mouse NIH-3T3 fibroblasts transfected with a retroviral vector expressing the 554-amino-acid product of a human 4-kilobase (kb) CSF-1 cDNA. Similar to results previously obtained with a 1.6-kb human cDNA that codes for a 256-amino-acid CSF-1 precursor, the results of the present study showed that NIH-3T3 cells expressing the product of the 4-kb clone produced biologically active human CSF-1 and were transformed by an autocrine mechanism when cotransfected with a vector containing a human c-fms (CSF-1 receptor) cDNA. The 4-kb CSF-1 cDNA product was synthesized as an integral transmembrane glycoprotein that was assembled into disulfide-linked dimers and rapidly underwent proteolytic cleavage to generate a soluble growth factor. Although the smaller CSF-1 precursor specified by the 1.6-kb human cDNA was stably expressed as a membrane-bound glycoprotein at the cell surface and was slowly cleaved to release the extracellular growth factor, the cell-associated product of the 4-kb clone was efficiently processed to the secreted form and was not detected on the plasma membrane. Digestion with glycosidic enzymes indicated that soluble CSF-1 encoded by the 4-kb cDNA contained both asparagine(N)-linked and O-linked carbohydrate chains, whereas the product of the 1.6-kb clone had only N-linked oligosaccharides. Removal of the carbohydrate indicated that the polypeptide chain of the secreted 4-kb cDNA product was longer than that of the corresponding form encoded by the smaller clone. These differences in posttranslational processing may reflect diverse physiological roles for the products of the two CSF-1 precursors in vivo.

Cell surface expression of v-fms-coded glycoproteins is required for transformation.

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 v-fms 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 proteolytically 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.

Ligand-induced tyrosine kinase activity of the colony-stimulating factor 1 receptor in a murine macrophage cell line.

Metabolic labeling of simian virus 40-immortalized murine macrophages with 32Pi and immunoblotting with antibodies to phosphotyrosine demonstrated that the c-fms proto-oncogene product (colony-stimulating factor 1 [CSF-1] receptor) was phosphorylated on tyrosine in vivo and rapidly degraded in response to CSF-1. Stimulation of the CSF-1 receptor also induced immediate phosphorylation of several other cellular proteins on tyrosine. By contrast, the mature cell surface glycoprotein encoded by the v-fms oncogene was phosphorylated on tyrosine in the absence of CSF-1, suggesting that it functions as a ligand-independent kinase.

The v-fms oncogene induces factor independence and tumorigenicity in CSF-1 dependent macrophage cell line

The McDonough strain of feline sarcoma virus (SM-FeSV) transforms fibroblast cell lines in culture and produces fibrosarcomas in domestic cats1,2. SM-FeSV does not induce haematopoietic malignancies in spite of the fact that its viral oncogene, v-fms, codes for a glycoprotein related to the receptor for the mononuclear phagocyte colony stimulating factor, CSF-1 (refs 3, 4). The v-fms-coded polypeptide includes the complete extracellular domain of the c-fms proto-oncogene product5,6 and retains the ability to bind CSF-1 specifically4. The two molecules have very similar sequences except at their extreme carboxyl terminal ends where 40 amino acids of the c-fms-coded glycoprotein are replaced by 11 unrelated residues in the v-fms product5. Autophosphorylation of the c-fms gene product on tyrosine is enhanced by CSF-1 addition3, whereas phosphorylation of the v-fms-coded glycoprotein appears to be constitutive4. We now show that introduction of the v-fms gene into simian virus-40 (SV40)-immortalized, CSF-1 dependent macrophages renders them independent of CSF-1 for growth and tumourigenic in nude mice. These factor-independent cell lines express unaltered levels of the c-fms product which is down-modulated in response to either CSF-1 or the tumour promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The induction of factor independence by a non-autocrine mechanism suggests that the v-fms product is an unregulated kinase that provides growth stimulatory signals in the absence of ligand.

Transmembrane orientation of glycoproteins encoded by the v-fms oncogene

The retroviral oncogene v-fms encodes a glycoprotein whose transport to the plasma membrane is required for transformation. Tryptic digestion of microsomes from transformed cells yielded membrane-protected amino-terminal fragments 40 kd smaller than intact molecules. These fragments were glycosylated, and they included v-fms-coded epitopes expressed at the cell surface. Deletion of the predicted membrane-spanning peptide generated polypeptides that were completely sequestered within microsomes. The mutant glycoproteins acquired more asparagine-linked oligosaccharide chains than did wild-type molecules, lacked kinase activity in vitro, were not transported to the cell surface, and had no transforming activity. Thus, the membrane-spanning segment in the middle of the glycoprotein interrupts translocation of nascent chains into the endoplasmic reticulum, ultimately orienting the amino-terminal domain outside the cell and the carboxy-terminal kinase domain in the cytoplasm. These topological features are similar to those of several growth factor receptors, suggesting that v-fms transforms cells through modified receptor-mediated signals.

The colony-stimulating factor 1 (CSF-1) receptor (c-fms proto- oncogene product) and its ligand

Alterations in genes that function in normal growth and development have been linked to malignant cell transformation. The mononuclear phagocyte colony-stimulating factor (CSF-1 or M-CSF) is a polypeptide growth factor synthesized by mesenchymal cells, which stimulates the survival, proliferation, and differentiation of haematopoietic cells of the monocyte-macrophage series. Multiple forms of soluble CSF-1 are produced by proteolytic cleavage of membrane-bound precursors, some of which are stably expressed at the cell surface. The c-fms proto-oncogene encodes the CSF-1 receptor, which is composed of an extracellular ligand-binding domain linked by a single membrane-spanning segment to a cytoplasmic tyrosine-specific protein kinase domain. Whereas the tyrosine kinase activity of the normal receptor is stimulated by CSF-1, mutations in the c-fms gene can constitutively activate the kinase to provide growth-stimulatory signals in the absence of the ligand. Oncogenic activation of the c-fms gene product appears to involve removal of a negative regulatory tyrosine residue near the carboxyl terminus of the receptor and one or more additional mutations that may simulate a conformational change induced by CSF-1 binding. Expression of the human c-fms gene in mouse NIH-3T3 cells confers a CSF-1 stimulated growth phenotype, indicating that receptor transduction is sufficient for fibroblasts to respond to a haematopoietic growth factor. In contrast, the v-fms oncogene induces factor-independent growth and tumorigenicity in factor-dependent myeloid cell lines, and contributes to the development of proliferative disorders of multiple haematopoietic lineages when introduced into murine bone marrow progenitors. Aberrant expression of an endogenous c-fms gene secondary to pro viral insertion and transcriptional activation has also been implicated in virus-induced myeloblastic leukaemia in mice. The c-fms and CSF-1 genes have been mapped on the long arm of human chromosome 5, a region that frequently undergoes interstitial deletions in certain haematopoietic disorders including acute myelogenous leukaemia. The study of CSF-1 and its receptor should provide information concerning the role of tyrosine kinases in regulating the normal growth and differentiation of haematopoietic cells and in contributing to their malignant transformation.

Biosynthesis of Macrophage Colony-Stimulating Factor (CSF-1): Differential Processing of CSF-1 Precursors Suggests Alternative Mechanisms for Stimulating CSF-1 Receptors

The macrophage colony-stimulating factor, M-CSF or CSF-1, is a glycosylated polypeptide homodimer required for the proliferation and differentiation of mononuclear phagocytic precursors and the survival of mature monocytes and macrophages (reviewed in Stanley et al., 1983). CSF-1 has also been reported to stimulate a variety of the immune effector functions of terminally differentiated macrophages such as phagocytosis and intracellular killing of microorganisms (Karbassi et al., 1987), tumor cell cytolysis (Wing et al., 1982; Ralph and Nakoinz, 1987), production of biocidal oxygen metabolites (Wing et al., 1985) and synthesis of plasminogen activator (Lin and Gordon 1979; Hamilton et al., 1980) as well as other cytokines including interleukin-1 (Moore et al., 1980), myeloid colony-stimulating activities (Metcalf and Nicola, 1985; Warren and Ralph, 1986), tumor necrosis factor (TNF) and interferon (Warren and Ralph, 1986). These diverse effects are mediated by binding of CSF-1 to a single high-affinity receptor on the plasma membrane of mononuclear phagocytes (Guilbert and Stanley, 1980 and 1986).

Antibodies to distal carboxyl terminal epitopes in the v-fms-coded glycoprotein do not cross-react with the c-fms gene product

The product of the v-fms oncogene is an integral transmembrane glycoprotein that is closely related to the cell surface receptor for the macrophage colony stimulating factor, CSF-1. A fragment of the v-fms gene encoding a major portion of the extracellular amino terminal domain, the membrane-spanning segment, and the entire carboxyl terminal tyrosine kinase domain of the glycoprotein was molecularly cloned into an inducible prokaryotic expression plasmid. Polypeptide products consisting only of v-fms-coded amino acids were produced in bacteria and were used to prepare immune reagents that precipitated the v-fms-coded glycoproteins expressed in transformed cells. Whereas rabbit antisera to recombinant polypeptides detected antigenic determinants of the c-fms proto-oncogene product, seven mouse monoclonal antibodies to these same antigens reacted only with v-fms-specific epitopes. Proteolytic mapping experiments and studies with a mutant v-fms-coded glycoprotein lacking the 37 carboxyl terminal amino acids of the wild-type product showed that the monoclonal antibodies were restricted in their reactivity to epitopes at the extreme carboxyl terminus of the glycoprotein. The v-fms and c-fms gene products must differ significantly in this region.