Identification of nonessential disulfide bonds and altered conformations in the v-sis protein, a homolog of the B chain of platelet-derived growth factor.

Abstract

The protein encoded by v-sis, the oncogene of simian sarcoma virus, is homologous to the B chain of platelet-derived growth factor (PDGF). There are eight conserved Cys residues between PDGF-B and the v-sis protein. Both native PDGF and the v-sis protein occur as disulfide-bonded dimers, probably containing both intramolecular and intermolecular disulfide bonds. Oligonucleotide-directed mutagenesis was used to change the Cys codons to Ser codons in the v-sis gene. Four single mutants lacked detectable biological activity, indicating that Cys-127, Cys-160, Cys-171, and Cys-208 are required for formation of a biologically active v-sis protein. The other four single mutants retained biological activity as determined in transformation assays, indicating that Cys-154, Cys-163, Cys-164, and Cys-210 are dispensable for biological activity. Double and triple mutants containing three of these altered sites were constructed, some of which were transforming as well. The v-sis proteins encoded by biologically active mutants displayed significantly reduced levels of dimeric protein compared with the wild-type v-sis protein, which dimerized very efficiently. Furthermore, a mutant with a termination codon at residue 209 exhibited partial transforming activity. This study thus suggests that the minimal region required for transformation consists of residues 127 to 208. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the v-sis proteins encoded by some of the biologically active mutants exhibited an altered conformation when compared with the wild-type v-sis protein, and suggested that Cys-154 and Cys-163 participate in a nonessential disulfide bond.

Mice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities.

“…1A). The tertiary structure of PDGF BB is dependent on intramolecular disulfide bridges, of which those between cysteine residues 1 and 6, as well as 3 and 7, respectively, have been shown to be necessary for the bioactivity of PDGF BB (Giese et al 1987;Sauer and Donoghue 1988;Ostman et al 1992;Heldin et al 1993). To generate a null mutation, we designed a targeting vector that would delete part of exon 3 and the entire exon 4 (Fig.…”

Dominant-negative mutants of a platelet-derived growth factor gene.

“…PDGF dimerization occurs through disulfide linkage between cysteine residues that are conserved between the A and B chains (Giese et al 1987;Sauer and Donoghue 1988), as well as between species at least as divergent as human and Xenopus (Mercola et al 1988). The mutation of any one of four distinct cysteines has been shown to abolish the transforming potential of \-sis (Giese et al 1987;Sauer and Donoghue 1988) and is presumed crucial to the formation of an active dimer.…”

“…(Giese et al 1987;Sauer and Donoghue 1988). The two mutant PDGF cDNA clones are indicated below, with the wild-type sequence indicated above the changes.…”

“…The mutation of any one of four distinct cysteines has been shown to abolish the transforming potential of \-sis (Giese et al 1987;Sauer and Donoghue 1988) and is presumed crucial to the formation of an active dimer. Because of the similarity between the PDGF isoforms, mutations at these crucial cysteines might perturb function in PDGF-A as well as w-sis.…”

The PDGFC CUB Domain Enhances Survival in PDGFC Mutant Mice

“…The presumed pdgf-c protein that is translated from pdgfc tm1lex should not have any GFD function, as paralogs of PDGF-C, PDGF-A and PDGF-B, require cysteines for dimerization and intramolecular and intermolecular folding [14,15]. We hypothesized that the pdgfc tm1lex protein product (pdgf-c mut ), lacking 6 of the 8 conserved cysteines, would have compromised or absent GFD signal transduction activity.…”