Using the BPV E5 protein as a model, we have devised a genetic approach that allows us to construct and identify small proteins with randomized transmembrane domains that can transform cells by inducing dimerization of growth factor receptors. We used a PCR-based method to construct complex retrovirus libraries in which the central transmembrane domain of the E5 protein is replaced with random hydrophobic amino acids. Isolates that induce cell transformation were selected on the basis of their ability to induce focus formation in rodent fibroblasts (Figure TM.1). We found that a surprisingly large fraction of random transmembrane domains can support cell transformation by interacting with the PDGF ß receptor. Some of these proteins display altered specificity compared to the wild-type E5 protein in terms of their ability to recognize PDGF ß receptor molecules with amino acid substitutions in the transmembrane domain. We believe that this approach can be extended to identify constructed small transmembrane proteins that activate or inhibit various cellular and viral transmembrane proteins. Because 30% of all cell proteins are integral membrane proteins, there are many potential targets of this approach. We are currently focusing on identifying small transmembrane proteins that activate other growth factor receptors, influence the resistance of cells to chemotherapeutic drugs, or inhibit infection by lytic viruses.

Figure TM.1 Scheme to isolate small transforming proteins with randomized transmembrane domains.