A Small v-sis/Platelet-Derived Growth Factor (PDGF) B-Protein Domain in Which Subtle Conformational Changes Abrogate PDGF Receptor Interaction and Transforming Activity

Abstract

Deletion scanning mutagenesis within the transforming region of the v-sis oncogene was used to dissect structure-function relationships. Mutations affecting codons within a domain encoding amino acids 136 through 148 had no effect upon homodimer formation or recognition by antisera which detect determinants dependent upon native intrachain disulfide linkages, yet the same mutations completely abolished transforming activity. A platelet-derived growth factor B (PDGF B) monoclonal antibody that prevents its interaction with PDGF receptors recognized v-sis, delta 142 (deletion of codon 142), and delta 148 but not delta 136, delta 137, or delta 139 mutants. These findings mapped the epitope recognized by this monoclonal antibody to include amino acid residues 136 to 139. Furthermore, mutations in the codon 136 to 148 domain caused markedly impaired ability to induce PDGF receptor tyrosine phosphorylation. Thus, subtle conformational alterations in this small domain critically affect PDGF receptor recognition and/or functional activation.

Platelet-derived growth factor AB heterodimer interchain interactions influence secretion as well as receptor binding and activation

“…We previously showed that truncation of the PDGF B carboxyl terminus at or beyond amino acid 185 results in efficient PDGF B secretion (LaRochelle et al, 1991). Thus we generated heterodimers by coexpressing PDGF A with wt PDGF B stop 185 or with cither of two mutants, PDGF B A109 and PDGF B A112, previously shown to exhibit substantially impaired transforming ability as homodimers (Giese et al, 1990). Furthermore, deletions of these single amino acid residues critically affected PDGFR recognition and functional activation (Giese et al, 1990).…”

“…There is evidence which supports the concept that PDGF interacts initially with one receptor and that such interactions affect subsequent recruitment of a second receptor (Bishayee etal., 1989;Fleidaran etal., 1991). Molecular genetic analysis has already identified at least two and possibly three independent sites in the PDGF B molecule that specify highaffinity interaction with the 0 PDGFR (LaRochelle et al, 1990(LaRochelle et al, , 1992Giese et al, 1990;Ostman et al, 1991b;Maher et al, 1993). PDGF A and B sites of interaction with the a PDGFR have yet to be elucidated.…”

“…MMTgpt was constructed for these studies by substitution of the gpt gene for the neomycin resistance gene of MMTneo at common EcoRI sites. PDGF B stop 185 (LaRochelle et al, 1991) and PDGF B deletion mutants (Giese et al, 1990) were previously described. PDGF B A109 and PDGF B A112 are analogous to deletions of amino acid residues 139 and 142, respectively, within the nascent v-sis gene product.…”

“…To produce the PDGF AB heterodimer in quantities sufficient for purification from the conditioned medium of NIH 3T3 transfectants, a secretory form of PDGF B, PDGF Bstop 185 (LaRochelle etal., 1991), was utilized. PDGF BA mutant (Giese et al, 1990) cDNAs were also expressed with a stop codon introduced at position 185. To determine the optimal conditions for PDGF AB stop 185 heterodimer formation, the MMTgpt PDGF A construct was titered in a cotransfection assay with 5 ug of the MMTneo PDGF B stop 185.…”