The platelet-derived growth factor beta receptor/bovine papillomavirus E5 connection: A model for transmembrane protein-protein interactions

The platelet derived growth factor dimer activates its receptor via extracellular binding, receptor dimerization, and autophosphorylation of cytoplasmic tyrosine residues. The activated receptor phosphorylates protein substrates, initiating a proliferative signal. The bovine papillomavirus E5 protein activates the platelet-derived growth factor β receptor (PDGFβR) tyrosine kinase independent of ligand. Evidence suggests that this 44-amino acid transmembrane dimer induces PDGFβR dimerization and activation via physical interactions between transmembrane amino acids of each protein. Transmembrane residues Gln17 and Thr513 of E5 and PDGFβR, respectively, as well as juxtamembrane Asp33 in E5 and an analogously positioned Lys499 in the PDGFβR have previously been found to be required for the PDGFβR/E5 interaction. The nature of this interaction was further characterized by additional mutagenesis of the PDGFβR transmembrane domain. We showed that dual conservative Lys499Arg/Thr513Ser substitutions in the PDGFβR are tolerated for an interaction with E5. We then showed that the PDGFβR/E5 interaction was disrupted when Thr513 was shifted to the carboxyl adjacent position, suggesting that Thr positioning within in the PDGFβR transmembrane α-helix plays a pivotal role in this interaction. Substitution of a Thr at position 513 in a receptor chimera retaining Lys499 but unable to interact with E5 appeared to inconsistently restore the interaction, implying that besides Lys499 and Thr513, other transmembrane amino acids of the receptor are required for an interaction with E5. Indeed, we found that the transmembrane residue Ile506 is significant in the interaction. Interestingly, Lys499, Ile506, and Thr513 of the receptor align along the same face of a predicted transmembrane alpha-helical configuration. This supports the model that the PDGFβR interacts with E5 via three direct protein-protein contacts along a single helical interface; involving a possible juxtamembrane electrostatic interaction and two transmembrane hydrogen bonds. We also identified roles for three receptor residues in the extracellular proximal transmembrane region. Global substitution studies involving PDGFβR transmembrane segments indicated that additional requirements remain in the carboxyl half of the transmembrane domain. These data suggest that an optimal PDGFβR/E5 interaction requires multiple transmembrane requirements, possibly involved in direct protein-protein contacts or in maintaining proper transmembrane helix conformation for optimal positioning of primary residue requirements.