Artificial Transmembrane Oncoproteins Smaller than the Bovine Papillomavirus E5 Protein Redefine Sequence Requirements for Activation of the Platelet-Derived Growth Factor (cid:2) Receptor (cid:1) †

The bovine papillomavirus E5 protein (BPV E5) is a 44-amino-acid homodimeric transmembrane protein that binds directly to the transmembrane domain of the platelet-derived growth factor (PDGF) (cid:1) receptor and induces ligand-independent receptor activation. Three specific features of BPV E5 are considered important for its ability to activate the PDGF (cid:1) receptor and transform mouse fibroblasts: a pair of C-terminal cysteines, a transmembrane glutamine, and a juxtamembrane aspartic acid. By using a new genetic technique to screen libraries expressing artificial transmembrane proteins for activators of the PDGF (cid:1) receptor, we isolated much smaller proteins, from 32 to 36 residues, that lack all filtered the retroviral supernatants through 0.45- (cid:5) m filters (Millipore, Leiden, The Netherlands), and concentrated the retrovirus in Amicon ultrahigh-speed centrifugal filtration con-centrators (Millipore). We infected parental BaF3 cells with the concentrated retrovirus as follows. We infected 5 (cid:6) 10 5 BaF3 cells in 500 (cid:5) l of medium per well of a 12-well dish with 500 (cid:5) l of concentrated virus. We added polybrene at a concentration of 4 (cid:5) g/ml and incubated the mixture at 37°C for 4 h. The infected cells were then transferred to a T25 flask containing 9 ml of RPMI/IL-3 medium with polybrene. After 48 h, we added G418 at a final concentration of 1 mg/ml. We subcloned the pool of infected, G418-resistant cells by serial dilution in a 96-well plate. We expanded individual clonal lines and tested them for receptor expression, either by immunoblotting (described below) or by de- termining their ability to survive in the absence of IL-3 and the presence of PDGF-BB (Calbiochem, San Diego, CA). Clonal lines expressing the desired receptor and maintaining a strict requirement for IL-3 were used for the experiments reported here. To express small transmembrane proteins in BaF3 cell lines, we prepared retroviral particles from HEK293T cells and infected BaF3 cells with up to 500 (cid:5) l of concentrated virus, as described above. To select for infected cells, the cells were incubated in RPMI/IL-3 medium plus 1 mg/ml hygromycin. We maintained selected cells in hygromycin and G418 (where applicable) at the concentrations used for selection. C127 cells at approximately 50% confluence in a 60-mm dish were infected as follows. Filtered, concentrated virus was brought to a volume of 1 ml in DMEM10. C127 cells were infected with a 1:20 dilution of this stock in DMEM10 to a final volume of 1 ml (virus