Alanine mutagenesis of conserved residues in the platelet-derived growth factor family: identification of residues necessary for dimerization and transformation.
Platelet-derived growth factor (PDGF) and vascular endothelial growth factor define a family of dimeric proteins characterized by eight conserved cysteine residues involved in disulfide bonds. Thirteen non-cysteine residues conserved among the platelet-derived/vascular endothelial growth factors were individually mutated to alanine in v-sis/PDGF-B. In addition, five other residues flanking F148 were also mutated to alanine. The resulting mutants were assayed for transformation of NIH3T3 cells, and the mutant proteins were assayed for their ability to dimerize. Four residues were found to be crucial for disulfide-linked dimer formation: P152 and G162 were mandatory, while R159 and H205 also contributed to efficient dimerization. Four of the mutant proteins (at residues N147, F148, L149 and K185) dimerized efficiently yet exhibited less than 50% transforming activity compared with wild-type v-sis. Two mutants (at residues D142 and F148) were located in a region important for PDGF receptor interaction and were further studied with regard to secretion and PDGF receptor autophosphorylation. A series of substitutions at residue F148 revealed a strong preference for aromatic amino acids. One mutant from this series (F148G) dimerized but was completely inactive for transformation. This study thus identifies four residues in v-sis/PDGF-B important for dimerization and also identifies additional residues critical for full activation of PDGF receptors. The E5 oncoprotein encoded by bovine papillomavirus type I exhibits two short regions of amino acid similarity when compared with the minimal transforming region of v-sis/PDGF-B. Several of the v-sis mutants discussed in this work affect residues that are also present in the E5 oncoprotein, including F148, L149 and H205.
Maher
D W
DW
Department of Chemistry, University of California, San Diego, La Jolla 92093-0322.
Strawn
L M
LM
Donoghue
D J
DJ
D000409:Alanine / Q000235:genetics*; D000595:Amino Acid Sequence; D002471:Cell Transformation, Neoplastic; D017124:Conserved Sequence; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D020575:Oncogene Proteins v-sis; D009856:Oncogene Proteins, Viral / Q000737:chemistry; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity; D013329:Structure-Activity Relationship
D020575:Oncogene Proteins v-sis; D009856:Oncogene Proteins, Viral; D010982:Platelet-Derived Growth Factor; D015682:Retroviridae Proteins, Oncogenic; C054304:oncogene protein E5, Bovine papillomavirus type 1; D017479:Receptors, Platelet-Derived Growth Factor; D000409:Alanine