The transmembrane domain of the E5 oncoprotein contains functionally discrete helical faces

The E5 protein of bovine papillomavirus is a 44-amino acid, Golgi-resident, type II transmembrane protein that efficiently transforms immortalized mou se fibroblasts. The transmembrane (TM) domain of E5 is not only critical fo r biological activity, it also regulates interactions with cellular targets including the platelet derived growth factor receptor (PDGF-R) and the 16- kDa subunit of the vacuolar proton ATPase (V-ATPase), In order to define th e specific TM amino acids essential for E5 biological and biochemical activ ity, we performed scanning alanine mutagenesis on 25 of the 30 potential TR I residues and genetically mapped discrete Lu-helical domains which separat ely regulated the ability of E5 to bind PDGF-R, activate PDGF-R, and to for m oligomers. Alanine substitutions at positions 17, 21, and 24 (which lie o n the same helical face) greatly inhibited E5 association with the PDGF-R, suggesting that this region comprises the receptor binding site. PDGF-R act ivation also mapped to a specific but broader domain in E5; mutant proteins with alanines on one helical face (positions 8, 9, 11, 16, 19, 22, and 23) continued to induce PDGF-R tyrosine phosphorylation, whereas mutant protei ns with alanines on the opposite helical face (positions 7, 10, 13, 17, 18, 21, 24, and 25) did not, indicating that the latter helical face was criti cal for mediating receptor transphosphorylation. Interestingly, these "acti vation-defective" mutants segregated into two classes: 1) those that were u nable to form dimers but that could still form higher order oligomers and t ransform cells, and 2) those that were defective for PDGF-R binding and wer e transformation-incompetent. These findings suggest that the ability of E5 to dimerize and to bind PDGF-R is important for receptor activation, Howev er, since several transformation-competent E5 mutants were defective for bi nding and/or activating PDGF-R, it is apparent that E5 must have additional activities to mediate cell transformation. Finally, alanine substitutions also defined two separate helical faces critical for E5/E5 interactions (ho modimer formation). Thus, our data identify distinct E5 helical faces that regulate homologous and heterologous intramembrane interactions and define two new classes of biologically active TM mutants.