Transformation-specific interaction of the bovine papillomavirus E5 oncoprotein with the platelet-derived growth factor receptor transmembrane domain and the epidermal growth factor receptor cytoplasmic domain

Abstract

The bovine papillomavirus ES transforming protein appears to activate both the epidermal growth factor receptor (EGF-R) and the platelet-derived growth factor receptor (PDGF-R) by a ligand-independent mechanism. To further investigate the ability of E5 to activate receptors of different classes and to determine whether this stimulation occurs through the extracellular domain required for ligand activation, we constructed chimeric genes encoding PDGF-R and EGF-R by interchanging the extracellular, membrane, and cytoplasmic coding domains. Chimeras were transfected into NIH 3T3 and CHO(LR73) cells. All chimeras expressed stable protein which, upon addition of the appropriate ligand, could be activated as assayed by tyrosine autophosphorylation and biological transformation. Cotransfection of E5 with the wild-type and chimeric receptors resulted in the ligand-independent activation of receptors, provided that a receptor contained either the transmembrane domain of the PDGF-R or the cytoplasmic domain of the EGF-R. Chimeric receptors that contained both of these domains exhibited the highest level of E5-induced biochemical and biological stimulation. These results imply that ES activates the PDGF-R and EGR-R by two distinct mechanisms, neither of which specifically involves the extracellular domain of the receptor. Consistent with the biochemical and biological activation data, coimmunoprecipitation studies demonstrated that ES formed a complex with any chimera that contained a PDGF-R transmembrane domain or an EGF-R cytoplasmic domain, with those chimeras containing both domains demonstrating the greatest efficiency of complex formation. These results suggest that although different domains of the PDGF-R and EGF-R are required for ES activation, both receptors are activated directly by formation of an E5-containing complex.

E5 oncoprotein retained in the endoplasmic reticulum/cis Golgi still induces PDGF receptor autophosphorylation but does not transform cells.

“…complex with PDGF-R (Goldstein et al, 1992a(Goldstein et al, , 1994DiMaio, 1992, 1994;Nilson and DiMaio, 1993) which is apparently mediated by specific interactions with the receptor TM domain (Goldstein et al, 1992a;Cohen et al, 1993a) rather than by its ligand binding domain (Petti and DiMaio, 1992).…”

“…Functionally, E5 can co-operate with the epidermal growth factor receptor (EGF-R) and colony stimulating factor-I receptor for transforming fibroblasts, and can induce the hyperphosphorylation/activation of EGF-R by a ligandindependent mechanism (Martin et al, 1989). E5 can also physically associate with EGF-R (Cohen et al, 1993a). However, in most cases, cell transformation by E5 appears to require the presence of the platelet-derived growth factor receptor (PDGF-R) (Petti et al, 1991;DiMaio, 1992, 1994;Nilson and DiMaio, 1993;Goldstein et al, 1994).…”

“…However, in most cases, cell transformation by E5 appears to require the presence of the platelet-derived growth factor receptor (PDGF-R) (Petti et al, 1991;DiMaio, 1992, 1994;Nilson and DiMaio, 1993;Goldstein et al, 1994). E5 binds and induces autophosphorylation of PDGF-R specifically (Petti et al, 1991;Petti and DiMaio, 1992) and forms a ternary complex with PDGF-R and the 16K subunit of the vacuolar H+-ATPase (V-ATPase) which is mediated by the TM domains of these proteins (Goldstein et al, 1992a;Cohen et al, 1993a). E5 does not appear to bind to PDGF-R by mimicry of PDGF , as previously suggested (Petti and DiMaio, 1992).…”

Structural models of the bovine papillomavirus E5 protein

“…Mutational analysis has identified specific residues in the transmembrane and juxtamembrane domains of both the E5 protein and the PDGF-␤ receptor that are required for complex formation and transformation, suggesting that these two proteins contact one another directly. [13][14][15][16][17][18] In contrast, the extracellular ligand binding domain of the receptor is not required for complex formation with the E5 protein, E5-induced receptor activation, or for mitogenic signaling. 6 The N-terminal 32 amino acids of the E5 protein are largely hydrophobic and are thought to form a transmembrane ␣-helix.…”

Beyond Channel Activity: Protein-Protein Interactions Involving Viroporins

“…Deletion of the extracellular ligand-binding domain of PDGFβR did not prevent its cooperation with E5, demonstrating that E5 activation of PDGFβR is ligand independent (Drummond-Barbosa et al 1995). Also, mutagenesis and chimeric studies of PDGFβR have mapped the binding region to the TMD of the receptor (Cohen et al 1993;Nappi et al 2002). The interaction between E5 and PDGFβR is also highly specific, since cooperation was not observed between E5 and PDGFαR, a closely related receptor tyrosine kinase (Goldstein et al 1994).…”