The bovine papillomavirus E5 protein and the PDGF beta receptor: it takes two to tango

Abstract

The extremely hydrophobic, 44-amino acid bovine papillomavirus (BPV) E5 protein is the smallest known oncoprotein, which orchestrates cell transformation by causing ligand-independent activation of a cellular receptor tyrosine kinase, the platelet-derived growth factor beta receptor (PDGFbetaR). The E5 protein forms a dimer in transformed cells and is essentially an isolated membrane-spanning segment that binds directly to the transmembrane domain of the PDGFbetaR, inducing receptor dimerization, autophosphorylation, and sustained mitogenic signaling. There are few sequence constraints for activity as long as the overall hydrophobicity of the E5 protein and its ability to dimerize are preserved. Nevertheless, the E5 protein is highly specific for the PDGFbetaR and does not activate other cellular proteins. Genetic screens of thousands of small, artificial hydrophobic proteins with randomized transmembrane domains inserted into an E5 scaffold identified proteins with diverse transmembrane sequences that activate the PDGFbetaR, including some activators as small as 32-amino acids. Analysis of these novel proteins has provided new insight into the requirements for PDGFbetaR activation and specific transmembrane recognition in general. These results suggest that small, transmembrane proteins can be constructed and selected that specifically bind to other cellular or viral transmembrane target proteins. By using this approach, we have isolated a 44-amino acid artificial transmembrane protein that appears to activate the human erythropoietin receptor. Studies of the tiny, hydrophobic BPV E5 protein have not only revealed a novel mechanism of viral oncogenesis, but have also suggested that it may be possible to develop artificial small proteins that specifically modulate much larger target proteins by acting within cellular or viral membranes.

Figure 1. Predicted amino acid sequence of the BPV E5 protein (top) and proposed transmembrane orientation of the E5 dimer (bottom)

Figure 2. The E5 protein is the major BPV oncogene in mouse fibroblasts

Mouse C127 cells were transfected with no viral DNA, wild-type BPV DNA, and a BPV mutant with a frameshift mutation that disrupts the E5 gene. Cells were stained after two weeks to demonstrate the appearance of transformed foci.

Figure 3. Activation of the PDGFβR by the E5 protein

BaF3 cells expressing the wild-type PDGFβR, a kinase-defective receptor mutant, and a receptor truncation mutant lacking most of the extracellular ligand binding domain were co-expressed in cells with the E5 protein (+) or an empty vector (-). Tyrosine phosphorylated PDGFβR was detected by immunoprecipitation followed by Western blotting for phosphotyrosine. M, p, and t indicate the position of the mature, precursor, and truncated forms of the PDGFβR, respectively. Adapted by permission from Macmillan Publishers Ltd: Oncogene 20:7866-7873, 2001.

Figure 4. The E5 protein and the PDGFR form a stable complex in cells

C127 cells expressing the wild-type E5 protein or the indicated E5 mutants were immunoprecipitated by an antibody that recognizes the E5 protein and immunoblotted with an antibody that recognizes the mature and precursor forms of the PDGFβR. Note that the wild-type PDGFβR and several receptor mutants associate with the E5 protein, but mutations at gln17, asp33, and the C-terminal cysteines prevent complex formation. (J. Virology (1995) 69:5869-5874, reproduced with permission from American Society for Microbiology.)

Figure 5. Schematic diagram of the complex between the E5 dimer and two molecules of the PDGFβR

The two white parallelograms represent the inner and outer leaflets of the membrane. The solid line represents the disulfide bonds that stabilize the E5 dimer, and the dotted lines represent salt bridges and hydrogen bonds mediating the association between the E5 protein and the PDGFβR, as well as a hydrogen bond between the glutamines that stabilizes the E5 dimer. C, cysteine; D, aspartic acid 33; K, lysine 499; Q, glutamine 17; T, threonine 513.

Figure 6. Surface representation of the E5 dimer as determined by molecular modeling

One face of the E5 dimer is shown. One E5 monomer in the dimer is colored dark blue and the other red. The essential aspartic acid (pink) and glutamine (light blue) were contributed by different monomers on one face of the dimer are shown. Figure courtesy of Brad Stanley and Donald Engelman.

Figure 7. Scheme to isolate small, transmembrane proteins with transforming activity from complex libraries

See text for details. Reprinted from J. Mol. Biol. 338, Freeman-Cook, et al., Selection and Characterization of Small Random Transmembrane Proteins that Bind and Activate the Platelet-derived Growth Factor β Receptor, pp. 907-920 (2004), with permission from Elsevier.

Figure 8. Diverse, artificial transmembrane proteins can activate the PDGFβR and transform cells

The figure shows the sequences of the transmembrane domain of small, transmembrane proteins that transform cells. These proteins were recovered from libraries in which a 15-amino acid segment was randomized to primarily hydrophobic amino acids, but the glutamine at position 17 (bold Q) was not varied. The top line shows the transmembrane sequence of the wild-type E5 protein. Reprinted from J. Mol. Biol. 338, Freeman-Cook, et al., Selection and Characterization of Small Random Transmembrane Proteins that Bind and Activate the Platelet-derived Growth Factor β Receptor, pp. 907-920 (2004), with permission from Elsevier.