Point mutations in dimerization motifs of transmembrane domain stabilize active or inactive state of the EphA2 receptor tyrosine kinase [Signal Transduction]

April 14th, 2014 by Sharonov, G. V., Bocharov, E. V., Kolosov, P. M., Astapova, M. V., Arseniev, A. S., Feofanov, A. V.

EphA2 receptor tyrosine kinase plays a central role in regulation of cell adhesion and guidance in many human tissues. Activation of EphA2 befalls after proper dimerization/ oligomerization in the plasma membrane, which occurs with participation of extracellular and cytoplasmic domains. Our recent studies revealed that isolated transmembrane domain (TMD) of EphA2 embedded into lipid bicelle dimerized via heptad repeat motif L535X3G539X2A542X3V546X2L549, rather than through alternative glycine zipper motif A536X3G540X3G544 (typical for TMD dimerization in many proteins). To evaluate 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 (wild type (WT), HR and GZ variants) in HEK293T cells. Confocal microscopy revealed similar distribution of all EphA2-YFP variants in cells. Expression of EphA2-YFP variants, their kinase activity (phosphorylation of Tyr588 and/or Tyr594) and ephrin-A3 binding were analyzed with a flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected for mutant variants. Mutations in TMD have significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for HR variant and reduced for GZ variant as compared to 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.
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