Understanding Periperal Membrane Protein Interactions | BPS Thematic Meeting

Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Monday Speaker Abstracts

BIOPHYSICS OF GROWTH FACTOR RECEPTOR SIGNALING IN THE MEMBRANE Kalina Hristova ; 1 Johns Hopkins University, Baltimore, MD, USA Microscopic GM1-enriched domains can form, in the plasma membrane of live mammalian cells expressing the EphA2 receptor tyrosine kinase, in response to its ligand ephrinA1-Fc. The GM1 enriched microdomains form concomitantly with EphA2-enriched microdomains. To gain insight into how plasma membrane heterogeneity controls signaling, we quantified the degree of EphA2 segregation and we studied the initial EphA2 signaling steps in both EphA2-enriched and EphA2-depleted domains using Forster Resonance Energy transfer and Number and Brightness. By measuring dissociation constants, we demonstrate that the propensity of EphA2 to oligomerize is similar in EphA2-enriched and -depleted domains. However, EphA2 interacts preferentially with its downstream effector SRC in EphA2-depleted domains. Given the critical role of EphA2 organization for signaling, we are using MINFLUX imaging as we seek detailed views of the EphA2-enriched and -depleted domains and mechanistic insights into oligomerization and domain formation. ANIONIC LIPIDS INHIBIT THE CATALYTIC ACTIVITY OF THE MEMBRANE PROXIMAL EGFR KINASE DOMAIN Adam W Smith 1 ; Ronald Villaber 1 ; 1 Texas Tech University, Chemistry & Biochemistry, Lubbock, TX, USA The epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase whose activity is regulated not only by ligand binding and dimerization, but also by its local membrane environment. While the role of lipids in EGFR clustering and signaling has been inferred from cell-based and computational studies, direct biochemical evidence for lipid-mediated regulation of EGFR catalytic activity has been lacking. Here, we reconstitute a catalytically active intracellular domain of EGFR on synthetic liposomes, enabling us to isolate the effects of specific lipid species on kinase activity. We show that both phosphatidylinositol 4,5 bisphosphate (PIP 2 ) and phosphatidylserine (PS) act as reversible, concentration-dependent inhibitors of EGFR activity, exhibiting mixed-mode inhibition with a predominant noncompetitive component. These findings suggest that anionic phospholipids can stabilize inactive conformations of the kinase domain through electrostatic interactions, potentially contributing to autoinhibition in the absence of ligand. Our results establish a minimal, membrane-based system for dissecting lipid-protein interactions that regulate EGFR and other peripheral membrane proteins. This work has the potential to resolve the chemical details of cell signaling and to improve therapeutic targeting strategies.

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