Our experiments determined the effect of all possible amino acids at position 17 on the ability of the E5 protein to bind to and activate the PDGF β receptor and to transform cells. In both C127 fibroblasts and Ba/F3 cells, all of the E5 mutant proteins that transformed cells formed complexes with the receptor and induced receptor autophosphorylation, whereas the transformation-defective mutant proteins did not efficiently bind or activate the receptor. The wild-type and transformation-competent mutant E5 proteins induced growth factor independence in Ba/F3 cells only when coexpressed with exogenous PDGF β receptor. Furthermore, a PDGF receptor-specific kinase inhibitor reduced receptor tyrosine phosphorylation, led to reversion of the transformed phenotype in C127 cells, and prevented IL-3-independent growth in Ba/F3 cells. Thus, the wild-type and transformation-competent mutant E5 proteins require the presence and sustained activation of the PDGF β receptor to transform cells. These results confirm the importance of position 17 of the E5 protein in PDGF β receptor binding and activation, imply that complex formation with the E5 protein is required for receptor activation, and provide compelling evidence that the PDGF β receptor is the primary target of the E5 protein.
The E5 protein also binds to a 16-kDa transmembrane subunit of the H+
-ATPase in some cell types (e.g., see reference 10
). We and others have not been able to detect this interaction in C127 cells (28
). Furthermore, in mouse NIH 3T3 cells, the ability of mutant E5 proteins to bind this protein shows no apparent correlation with transforming efficiency and appears to require only a positive charge at position 17 and a negative charge on the ATPase subunit (28
These experiments extend the work of two other groups. Meyer et al. (20
) examined the focus-forming efficiencies of several position 17 mutants in NIH 3T3 cells. Since transformation appeared to require a residue at position 17 capable of forming hydrogen bonds, they speculated that the main function of glutamine 17 was to form interhelical hydrogen bonds to stabilize the E5 dimer. However, E5 expression and dimerization by these mutants were not assessed, nor were PDGF β receptor binding and activation examined. Sparkowski et al. analyzed a panel of position 17 mutants in NIH 3T3 and C127 cells and reported that most of the transformation-competent mutants induced PDGF β receptor phosphorylation without binding the receptor and that the serine mutant transformed cells without inducing PDGF receptor activation (27
). In addition, they saw no effect of the position 17 mutations on E5 dimerization. Several factors are likely to account for the differences from our results. First, the basal level of phosphorylation of the mature form of the receptor was high in the experiments of Sparkowski and colleagues, precluding further analysis of its phosphorylation and focusing their attention on the immature form of the receptor (28
). However, in our experiments, the active position 17 mutant E5 proteins were impaired in their ability to bind and induce autophosphorylation of the immature form of the receptor, so conclusions about the activities of various mutant E5 proteins cannot be based on study of this receptor form alone. The basis for this impaired interaction of the mutants with the precursor form of the receptor is not known. Second, we found that the stringent buffer RIPA disrupts complexes containing most transformation-competent mutants, whereas the gentle lysis buffer CHAPS preserves these interactions. Thus, a possible explanation for the findings of Sparkowski et al. (28
) was the use of extraction buffers that disrupted complexes containing these mutants. Finally, they constructed their position 17 substitutions in a mutant, epitope-tagged version of the E5 protein (27
), whereas we analyzed the authentic E5 protein, differing from the wild-type protein only by the identity of the amino acid at position 17. It is possible that the presence of the epitope affected the properties of the E5 mutants.
In the experiments reported here, we constructed and analyzed all possible position 17 E5 mutants to gain a comprehensive picture of the role of the residue at this position. The residue at position 17 affected the ability of the E5 protein to dimerize and form stable complexes with the PDGF β receptor. Representative mutants localized normally in cells, and almost all of the mutant E5 proteins accumulated in cells, suggesting that the mutations did not affect appropriate partitioning of the E5 protein into the hydrophobic membrane environment. However, the transformation-defective arginine mutant was present in low levels. It is possible that the arginine side chain was unable to deprotonate to form the neutral species and partition into the membrane, resulting in decreased stability of this mutant.
Previous analyses of mutants with substitutions at the cysteines, which mediate E5 dimerization, imply that dimerization is required for complex formation and transformation (13
), and we have proposed that dimer formation is required to generate the binding site on the E5 protein for the PDGF β receptor (30
). The results reported here provide new insight into the role of dimerization in transformation and the role of position 17 in dimer formation. In general, the mutants with strongly polar residues at position 17 showed high levels of dimer, similar to those of the wild-type protein, whereas the residues with less-polar side chains, including serine and threonine, resulted in higher levels of monomer (Table ). However, all the position 17 mutants formed some dimers and there was no strict correlation between the extent of dimer formation and transforming activity. For example, the serine mutant, which transformed cells very efficiently, showed intermediate levels of dimer, similar to those of most nontransforming mutants. This shows that relatively low levels of dimer are sufficient for receptor binding and activation and suggests that the transformation defects of various mutants are not due to the inability of these mutants to dimerize efficiently. However, it is possible that the position 17 mutations caused local perturbations in the structure of the E5 dimer that affected receptor binding and activation.
The residue at position 17 may influence complex formation, and hence transformation, by directly affecting interactions that stabilize the E5-PDGF β receptor complex, as well as by affecting dimerization of the E5 protein. All of the mutants containing hydrophobic residues with side chains unable to form hydrogen bonds were defective for transformation, a result consistent with the proposed hydrogen bond between the residue at position 17 and threonine 513 in the transmembrane domain of the PDGF β receptor. The tyrosine, proline, and cysteine mutants, although capable of hydrogen bonding, were also defective for complex formation and transformation. Proline and tyrosine are largely excluded from the hydrophobic transmembrane region of proteins having single-membrane-spanning helices, and tyrosine occurs preferentially in the region of the polar headgroups in membrane proteins (18
). Proline disrupts the regular hydrogen bonding pattern along the helix backbone, causing kinks in transmembrane helices (31
), and tyrosine substitutions in the transmembrane domain of glycophorin A disrupted dimerization even when located at positions not in the helix interface (19
). Thus, tyrosine and proline may interfere with proper E5 dimerization or interaction with the PDGF β receptor.
Several polar amino acids at position 17 supported complex formation and transformation. Of these, glutamine and glutamic acid appeared to impart the most stability to the E5-receptor complex, based on its ability to withstand RIPA buffer extraction. This may be due to the length and strongly polar nature of these side chains which have great conformational flexibility and facilitate the formation of strong hydrogen bonds. Tryptophan and methionine, residues with substantial hydrophobic character, also allowed transformation. Tryptophan differs from the nontransforming hydrophobic residues in that it has an NH group on the side chain indole ring which is capable of hydrogen bonding. Similarly, methionine is able to hydrogen bond through its side chain sulfur atom (12
). For example, there is a hydrogen bond between a hydroxyl group of a threonine residue and a methionine sulfur in the crystal structure of myohemerythrin (11
). The aspartic acid mutant displayed intermediate transforming activity even though it bound the receptor well and, at least in some cell lines, induced a high level of receptor tyrosine phosphorylation. It is possible that this mutant induced the formation of aberrant complexes such as, for example, ones in which the sites of receptor phosphorylation differed from the sites in complexes containing the wild-type E5 protein.
Pairwise comparisons of mutants containing similar amino acid substitutions also highlight the importance of hydrogen bonds involving the side chain at position 17. For example, valine and threonine are isosteric β-branched amino acids. The threonine hydroxyl group and each valine side chain methyl group have roughly the same molecular volume, but the hydroxyl-group, unlike the methyl group, is capable of hydrogen bonding interactions. The valine mutant did not form complexes with the PDGF β receptor or lead to transformation, whereas the threonine mutant transformed cells far better than the valine mutant, formed complexes with the receptor, and induced intermediate levels of receptor tyrosine phosphorylation. The different activities of these mutants did not reflect differences in dimerization. Rather, these results strongly argue that hydrogen bonding by the amino acid at position 17 is an essential element in stabilizing the E5-receptor complex. Similarly, the serine mutant transformed at high efficiency, while the cysteine mutant was transformation defective. These different phenotypes do not appear related to dimerization efficiency and are likely to result from differences in the chemical properties of the cysteine sulfhydryl group and the serine hydroxyl group, such as the ability of the oxygen atom in the serine to form stronger hydrogen bonds than the cysteine sulfur atom (15
The effects of the mutations on dimerization suggest that the position 17 side chain forms contacts between E5 monomers, while the data on complex formation suggest that it forms contacts with the receptor. In both proposed conformations of the wild-type and representative transformation-competent E5 dimers, it was possible to dock aspartate 33 of the E5 protein with lysine 499 of the receptor and the residue at position 17 of the E5 protein with threonine 513 of the receptor. Therefore, these models can account for the ability of the residue at position 17 to influence both E5 dimer formation and complex formation with the PDGF receptor.
In summary, these studies provide a coherent view of the role of glutamine 17 in cell transformation by the E5 protein. This analysis of all possible substitutions at position 17 has revealed an excellent correlation between the ability of these mutants to form a stable complex with the PDGF β receptor, induce receptor activation, and cause transformation. The activities of the individual mutants and molecular modeling studies provide further evidence that the residue at position 17 contributes to transformation by stabilizing the E5 dimer and by interacting with threonine 513 in the PDGF β receptor transmembrane domain.