To clarify the role of aspartic acid 33 in E5 transformation and to test the hypothesis that there is an essential interaction between this residue and lysine 499 in the PDGF β receptor, we constructed and analyzed the effects of all possible substitutions at this position of the E5 protein. The glutamic acid mutant transformed cells approximately twice as well as the wild type, the proline mutant transformed approximately as well as the wild type, and four hydrophilic substitutions resulted in substantially lower but detectable transforming activity. The glutamic acid and proline mutants efficiently bound the PDGF β receptor and induced high levels of receptor tyrosine phosphorylation, while the four mutants with moderate transformation defects bound the receptor much less well than the wild-type E5 protein and induced lower levels of receptor phosphorylation. All other position 33 mutants failed to transform C127 cells and were significantly impaired in the ability to bind and activate the PDGF β receptor. In addition, a kinase inhibitor specific for the PDGF receptor reduced receptor tyrosine phosphorylation and led to reversion of the transformed phenotype in cells expressing the proline and glutamic acid mutants. These results highlighted the importance of the residue at position 33 of the E5 protein in cell transformation and binding and activation of the PDGF β receptor and provided further evidence that this receptor is the main target of the E5 protein in murine fibroblasts.
All of the mutant E5 proteins accumulated in cells, and representative mutants localized normally and formed dimers at levels similar to those for the wild-type protein. Therefore, altered stability, dimerization, or localization did not appear to be responsible for the phenotypes of the various mutants. It also seems unlikely that altered orientation of the E5 protein in the membrane was responsible for the behavior of the mutants. The difference in the charge of the N-terminal versus C-terminal juxtamembrane segment of single-span transmembrane proteins has been proposed to be the primary determinant of orientation (7
). However, transforming activity of the E5 mutants did not correlate in any simple way with juxtamembrane charge, since transformation was severely impaired by most neutral amino acids at position 33, even though other neutral amino acids (and lysine, a basic one) at this position allowed transformation. Furthermore, replacing the negative charge at position 33 with alanine inhibited transformation, whereas replacing the negative charge at position 36 did not inhibit. Other models propose that the sequence of N-terminal segment or the length of the hydrophobic domain are crucial for specifying orientation of type II membrane proteins (4
), but the mutations studied here did not affect either of these segments.
We previously showed that a positive charge in the extracytoplasmic juxtamembrane domain of the PDGF β receptor is required for a productive interaction between the E5 protein and the receptor (19
). Here we showed that a negative charge in the corresponding region of the E5 protein was also required for this interaction. Evidently, either aspartic acid or glutamic acid can function at position 33, since a negative charge at position 36 was not required when either of these acidic amino acids occupied position 33. Several amino acids without a negative charge, most notably proline, could also substitute for the wild type aspartic acid 33, but a negative charge at position 36 was required for the transforming activity of these position 33 mutants. Thus, a negative charge in the juxtamembrane region of the E5 protein and a positive charge in this region of the PDGF receptor are necessary for the productive interaction between these two proteins and for cell transformation. The simplest explanation for these results is that the E5 protein and the PDGF β receptor contact one another directly and that this complex is stabilized by a salt bridge between oppositely charged residues in the juxtamembrane region.
We have not been able to reconstitute this interaction by swapping the positive and negative charges on the E5 protein and the PDGF receptor. There are numerous possible explanations for the failure of these mutants to complement each other. For example, changing the sequence context of the juxtamembrane charges may alter their translational position relative to the negatively charged membrane surface, thereby preventing the interaction.
These experiments confirm and extend our earlier findings that mutations at position 33 impair C127 cell transformation and productive interaction with the PDGF β receptor (8
). Meyer et al. reported that the mutants D33A and E36A were both able to transform NIH 3T3 cells (14
); in our experiments the mutant D33A was defective for C127 cell transformation. It is possible that differences between the cell types and transformation assays used account for the difference observed in the activity of the D33A mutant. In addition, the D33A mutant used by Meyer et al. contained a second mutation, substituting a glutamic acid for glutamine at position 17 (14
). We showed previously that the Q17E mutation leads to an approximate doubling of transforming activity in C127 cells (10
), and it is possible that in their experiments the Q17E mutation compensated for the loss of the negative charge at position 33. In any event, Meyer et al. (14
) speculated that either aspartic acid 33 or glutamic acid 36 of the E5 protein interacted with the juxtamembrane lysine on the PDGF β receptor and concluded that the aspartic acid was probably more important than the glutamic acid, conclusions consistent with the biochemical analysis reported here. The importance of aspartic acid 33 compared to glutamic acid 36 is also suggested by the absolute conservation of aspartic acid 33 in the E5 proteins of all of the fibropapillomaviruses, in contrast to the absence of a negative charge at position 36 in the other E5 proteins, including the deer papillomavirus E5 protein, which also activates the PDGF β receptor and transforms C127 cells (11
The analysis of the double mutants suggests that the lysine on the PDGF β receptor interacted with glutamic acid 36 in the transformation-competent E5 mutants without a negative charge at position 33. Our previous spectroscopic analysis indicated that the E5 protein is largely α-helical and that the α-helical segment spans the membrane and includes aspartic acid 33 and possibly glutamic acid 36 as well (23
). Helical secondary structure in the juxtamembrane region of the wild-type E5 protein would place aspartic acid 33 and glutamic acid 36 on the same face of the helix (Fig. A), with the potential for the glutamic acid to contact the lysine on the receptor with only a modest change in the configuration of the E5 protein. Presumably, the transformation-competent mutants without a negative charge at position 33 assumed a conformation that steered the negatively charged residue at position 36 into position to interact with the lysine, whereas the defective position 33 mutants failed to do so. In contrast, if the E5 protein had β-sheet structure in this region, then aspartic acid 33 and glutamic acid 36 would not be in near alignment, and it would be more difficult to imagine how the glutamic acid 36 could substitute for the missing aspartic acid 33.
FIG. 9 Helical wheel diagrams of the E5 protein in a canonical α-helix (A), a right-handed coil-coil (B), and a left-handed coiled-coil (C). Since paired transmembrane helices typically pack in either right- or left-handed coiled-coil arrangements, the (more ...)
If aspartic acid 33 forms a salt bridge with the receptor, it must be oriented away from the E5 dimer interface. This is consistent with our result that the identity of the residue at position 33 did not influence E5 dimerization. In contrast, we previously demonstrated that some position 17 mutations had marked effects on dimerization, suggesting that glutamine 17 is at least partially buried in the dimer interface, where it can contribute to the stability of the E5 dimer (10
). These considerations suggest that aspartic acid 33 and glutamine 17 are situated on opposite faces of the E5 helix, the arrangement that would result if the E5 dimer exists as a left-handed coiled-coil (Fig. C). In contrast, a right-handed coiled-coil would place these two residues on the same face of the helix (Fig. B). Our spectroscopic data and the molecular modeling also predicted that the E5 dimer would assume a left-handed coiled-coil conformation (23
The data reported here strongly suggest the existence of a direct interaction between the juxtamembrane lysine on the PDGF β receptor and a negatively charged juxtamembrane residue on the E5 protein. We previously demonstrated that PDGF receptor binding and activation required a residue at position 17 of the E5 protein that can form hydrogen bonds, presumably with threonine 513 of the PDGF β receptor (10
). Finally, transforming activity displays relatively relaxed requirements for the precise sequence of hydrophobic residues in the transmembrane domain of the E5 protein (12
). Taken together, these and previous studies have identified the minimal requirements of the dimeric E5 protein for interaction with the PDGF β receptor and cell transformation: a hydrophobic transmembrane domain whose sequence may vary considerably, a hydrogen-bonding residue at position 17, and a juxtamembrane extracytoplasmic negative charge. It may be possible to design heterologous peptides incorporating these features which could bind the PDGF β receptor and perhaps serve as starting points for the design of peptides which could interact with and influence the activity of a variety of receptor tyrosine kinases. In addition, detailed characterization of the interaction between the E5 protein and the PDGF β receptor may reveal general principles governing assembly of transmembrane protein complexes.