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.

Abstract

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.

Cell specific transformation by c-fms activating loop mutations is attributable to constitutive receptor degradation

“…Figure 4a (lanes 1 ± 3) is a pulse-labelling study which demonstrates that most of the normal, immature 130 kD protein labelled in 30 min was converted to the mature form after a 90 min chase period ( Figure 4a, lane 2). The amount of mature form was reduced after 180 min chase (Figure 4a, lane 3), consistent with its known turnover rate in the absence of M-CSF (Rettenmier et al, 1987). Figure 4a, lanes 4 ± 6 demonstrate that the addition of M-CSF accelerated receptor degradation as expected (see Roth and Stanley, 1992).…”

The 230 kDa mature form of KDR/Flk-1 (VEGF receptor-2) activates the PLC-γ pathway and partially induces mitotic signals in NIH3T3 fibroblasts

“…The processing of CSF-1-R which belongs to the 5-Ig receptor and distantly related to the 7-Ig type Flt family was previously shown to be similar to those of KDR/Flk-1 described here (Rettenmier et al, 1987;Rohrschneider et al, 1989). This indicates that 5-Ig/7- Ig receptor tyrosine kinase supergene family utilizes a similar processing machinery.…”

Evidence that downregulation of the M-CSF receptor is not dependent upon receptor kinase activity

“…In the absence of M-CSF the mature form of the normal receptor evident in lane 1 (Figure 3a) disappeared with a half (Figure 3a and e and data not shown), indicating that the receptor was turned over in the absence of M-CSF, as expected (Rettenmier et al, 1987). The addition of M-CSF markedly enhanced its turnover, also as expected (Rettenmier et al, 1987). The degradation of the K616A mutant receptors was not enhanced by the addition of M-CSF although these receptors were still turned-over in its absence ( Figure 3b and f), as expected (Downing et al, 1989).…”

“…The receptors were labelled for 3 h with 35 S-methionine (Figure 3, lanes 1 and 6) and then chased with cold methionine for 1, 2, 3 and 4 h in the absence (lanes 2 ± 5) or presence of M-CSF (lanes 7 ± 10). In the absence of M-CSF the mature form of the normal receptor evident in lane 1 (Figure 3a) disappeared with a half (Figure 3a and e and data not shown), indicating that the receptor was turned over in the absence of M-CSF, as expected (Rettenmier et al, 1987). The addition of M-CSF markedly enhanced its turnover, also as expected (Rettenmier et al, 1987).…”