Biophysical Society Conference | Tahoe 2022

Molecular Biophysics of Membranes

Poster Abstracts

3-POS Board 1 PIP2 PROMOTES SELECTIVE DIMERIZATION OF THE MEMBRANE REGION OF THE EPHA2 RECEPTOR Katherine M Stefanski 1 ; Westerfield M Justin 1 ; Charles M Russell 1 ; Rajan Lamichhane 1 ; Francisco N. Barrera 1 ; 1 University of Tennessee, Knoxville, TN, USA The influence of the EphA2 receptor on cancer malignancy hinges on the two different ways it can be activated. EphA2 induces anti-oncogenic signaling after ligand binding. However, alternate activation of EphA2 by kinases in the absence of ligand generates a metastatic phenotype. The transmembrane (TM) domain of EphA2 adopts two alternate conformations in the ligand-dependent and the ligand-independent states. However, it is poorly understood how the different TM helical crossing angles that characterize the two states impact the activity and regulation of EphA2. We devised a method that uses bilayer hydrophobic matching to stabilize these two conformations for a sequence comprising the TM domain and a portion of the intracellular juxtamembrane (JM) segment. The two conformations have different TM crossing angles, as expected for the ligand-dependent and ligand-independent states of EphA2. We devised a single-molecule approach that uses SMALP native nanodiscs to measure dimerization in membranes. Using this method we observed that the signaling lipid PIP 2 (phosphatidylinositol 4,5-bisphosphate) promotes TM dimerization, but only in the conformation with a small crossing angle, which we propose corresponds to the ligand-independent state. In this conformation the two TM are almost parallel, and the positively charged JM segments are close to each other and cause electrostatic repulsion that reduces dimerization. We propose that that PIP 2 uses its high density of negative charges to alleviate JM repulsion, promoting EphA2 dimerization. The single-molecule results indicate a conformational coupling between the TM and JM regions of EphA2. Our work additionally suggests that PIP 2 directly exerts a regulatory effect on EphA2 activation in cells that is specific to the pro-oncogenic, ligand-independent state of the receptor. These data suggest that modulation of the lipid make-up around the EphA2 receptor could achieve an anti-tumor effect.

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