Point Mutations in Dimerization Motifs of the Transmembrane Domain Stabilize Active or Inactive State of the EphA2 Receptor Tyrosine Kinase

Abstract

Background: Isolated Eph transmembrane domains (TMD) dimerize in membrane mimetics, but the functional significance of these interactions is unclear. Results: Mutations introduced into the alternative dimerization motifs of the EphA2 TMD induced an opposite effect on receptor activity. Conclusion: Alternative TMD interactions promote either the active or inactive EphA2 conformation. Significance: The involvement of TMD interactions in Eph receptor activity is discovered.The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/ oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L 535 X 3 G 539 X 2 A 542 X 3 V 546 X 2 L 549 rather than through the alternative glycine zipper motif A 536 X 3 G 540 X 3 G 544(typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr 588 and/or Tyr 594) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.Receptor tyrosine kinases of the Eph family and their ephrin ligands are key regulators of cell-cell and cell-matrix adhesion, coordinating cell migration and positioning in various adult and embryonic tissues of human organisms (1, 2). The EphA2 receptor, a representative of the human 14-member Eph family, controls such diverse processes as capillary stabilization by pericytes (3), keratinocyte movement out of the basal layer (2), blastocyst entry into the endometrial layer (4), and cardiac stem cell mobilization from the niche (5). Activation of EphA2 leads to cell detachment (mobilization), loss of intercellular contacts (an increase in cell layer permeability), or cell repulsion (guidance).A classical model of EphA2 activation assumes the binding of a li...

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