The Bovine Papillomavirus E5 Protein Requires a Juxtamembrane Negative Charge for Activation of the Platelet-Derived Growth Factor β Receptor and Transformation of C127 Cells

The 44-amino-acid E5 protein of bovine papillomavirus type 1 (BPV) is the smallest known transforming protein (20). This homodimeric transmembrane protein, which is localized largely to membranes of the endoplasmic reticulum and Golgi apparatus, transforms fibroblasts by forming a stable complex with the endogenous platelet-derived growth factor (PDGF) β receptor and inducing ligand-independent receptor oligomerization and activation (1, 3, 6, 13, 17, 18, 23). The study of this unusual mechanism of receptor tyrosine kinase activation promises to lead to a greater understanding of both viral transformation and receptor biochemistry. Experiments with chimeric and mutant receptors showed that removal of the ligand-binding domain of the PDGF β receptor does not disrupt the interaction with the E5 protein and mapped the site of interaction between the E5 protein and the PDGF β receptor to the transmembrane/juxtamembrane regions of the two proteins (2, 3, 5, 19, 22). In contrast, PDGF induces receptor activation by binding to the extracellular domain of the receptor. Therefore, the interactions by which the E5 protein induces PDGF receptor activation must be strikingly different from those utilized by PDGF. On the basis of molecular modeling and infrared spectroscopy, we developed a model for the E5 dimer that consists of two long transmembrane helices that pack together in a left-handed coiled coil (23). We have proposed that the E5 dimer interacts directly with the transmembrane/juxtamembrane domains of two PDGF β receptor molecules, with both E5 monomers contributing to a binding site on each face of the E5 dimer. Thus, the E5 dimer is thought to serve as a transmembrane scaffold for dimerization of the PDGF β receptor, allowing the receptor to undergo trans phosphorylation and activation in the absence of PDGF (13, 23). The E5 protein can also form a stable complex with the hydrophobic 16-kDa subunit of the vacuolar H+-ATPase, but there is no compelling evidence that this interaction plays a role in PDGF receptor activation or fibroblast transformation (5, 21). The E5 protein consists of a hydrophobic N-terminal segment of 30 amino acids that spans membranes and a hydrophilic 14-amino-acid segment at the C terminus (20, 23). Four absolutely conserved residues in the E5 protein are important for binding and activation of the PDGF β receptor and for cell transformation: the transmembrane glutamine 17, the juxtamembrane aspartic acid 33, and the C-terminal cysteines 37 and 39, which are involved in homodimerization of the E5 protein (Fig. ​(Fig.1)1) (8, 10, 14, 15). In addition, the overall hydrophobicity of the central region of the E5 protein, but not the specific amino acid sequence, is critical for cell transformation (12, 14). FIG. 1 Alignment of the transmembrane sequences of the E5 protein and the PDGF β receptor. The E5 protein and transmembrane region of the PDGF β receptor (PDGFr) are shown in their antiparallel orientation, and the putative transmembrane region ... Mutational analysis also demonstrated that a positive charge in the extracellular juxtamembrane region of the PDGF β receptor and the transmembrane threonine 513 are required for interaction with the E5 protein and for E5-induced receptor activation but not for activation by PDGF (19). Since the E5 protein is thought to be inserted in the membrane in the orientation opposite that of the PDGF β receptor, aspartate 33 of the E5 protein and lysine 499 of the receptor both lie on the extracytoplasmic face of the membrane, with glutamine 17 of the E5 protein and threonine 513 of the receptor buried in the membrane at approximately the same distance from the membrane surface (Fig. ​(Fig.1).1). These data have led to the proposal that the E5 protein and the PDGF β receptor interact directly with one another and that two pairs of interacting residues, aspartate 33-lysine 499 and glutamine 17-threonine 513, stabilize the E5 protein-PDGF β receptor complex (10, 14, 19, 23). We began to test this proposal by analyzing a panel of E5 mutants containing every possible substitution at position 17 (10). There was an excellent correlation between the ability of these mutants to bind the receptor, induce receptor activation, and transform cells. All active E5 mutants contained a residue at position 17 that was capable of hydrogen bonding, consistent with the proposed hydrogen bond between glutamine 17 and threonine 513 of the receptor. Our previous mutational analysis of aspartic acid 33 showed that the mutation D33V abolished interaction with the PDGF β receptor and transformation of C127 fibroblasts and that the mutation D33N significantly reduced transformation (8, 15). Meyer et al. demonstrated that alanine substitutions at the negatively charged aspartic acid 33 and glutamic acid 36 inhibited transformation of NIH 3T3 cells (14). On the basis of that result, they speculated that the C-terminal aspartic acid 33 or glutamic acid 36 of the E5 protein interacted with the juxtamembrane lysine on the receptor. However, the ability of these mutants to bind to or induce activation of the PDGF β receptor was not determined, nor was the ability of amino acids other than alanine to functionally substitute for aspartic acid 33. In this study, we determined the functional range of amino acids at position 33 which allowed a productive interaction between the E5 protein and the PDGF β receptor, leading to cell transformation. We constructed and analyzed a panel of mutants containing all possible amino acids at position 33 of the E5 protein as well as several double mutants with mutations at both positions 33 and 36. These experiments revealed an absolute requirement for a juxtamembrane negative charge on the E5 protein for interaction with the PDGF β receptor and cell transformation.