The 44-amino acid bovine papillomavirus E5 protein is the smallest naturally-occurring oncoprotein, causing cell transformation by binding as a homodimer to the cellular receptor tyrosine kinase platelet-derived growth factor β (PDGF β) receptor and inducing receptor dimerization, trans-phosphorylation and mitogenic signaling. From a small transmembrane protein library, we isolated a 32-residue oncoprotein, pTM32-1, that activates the PDGF β receptor, inducing receptor phosphorylation and growth factor independence in Ba/F3-βR cells but not focus formation in C127 cells. Truncated, 33-amino acid ES exhibits the same growth phenotypes as pTM32-1. We screened a library of 36-residue randomized transmembrane proteins and identified two, pTM36-3 and pTM36-4, that induced growth factor independence in Ba/F3-13R cells and focus formation in C127 cells by activating the PDGF β receptor. These proteins lack all of the specific sequence features required for wild-type E5 to activate the PDGF β receptor, yet they exhibit similar growth phenotypes.

Peptide studies indicate that pTM36-4 forms a homodimer like the ES protein, and molecular modeling of pTM36-4 and another small, transmembrane activator of the PDGF β receptor, pTM45-5, identified a left-handed coiled coil structure that was common to both proteins. Alanine scanning mutagenesis along the transmembrane domain of pTM36-4 indicated that five of seven predicted homodimeric interface residues are required for focus formation, as is a transmembrane asparagine. The mutational data support the molecular modeling results, indicating that two small transmembrane proteins that differ in primary amino acid sequence but share the same function—namely transmembrane activation of PDGF β receptor—may also share the same structure.

Identification of pTM32-1 and pTM36-4 inspired synthesis of a library of 28-residue, predominantly randomized proteins that have no resemblance to the wild-type E5 protein. Screening this library in C127 cells will allow identification of tiny transmembrane proteins completely different from the ES protein that activate the PDGF β receptor or possibly other cellular membrane proteins. The tiny transmembrane oncoproteins described here bear little resemblance to the viral ES oncoprotein but can nevertheless productively interact with the same cellular target. We speculate that similar cellular proteins may exist but have been overlooked due to their small size and hydrophobicity.