Biophysical Society Conference | Tahoe 2024

Molecular Biophysics of Membranes

Thursday Speaker Abstracts

CHOLESTEROL CONTROLS THE ASSEMBLY AND ACTIVITY OF THE EPHA2 RECEPTOR Francisco N. Barrera 1 ; 1 University of Tennessee, Knoxville, TN, USA The receptor tyrosine kinase EphA2 plays a central role during embryogenic development of the neural system. EphA2 has low expression in adult tissues, with the exception of tumors, where it promotes malignancy in different cancer types, with a salient role in metastasis. At the molecular level, EphA2 can be found in different self-assembly states: as a monomer, dimer and oligomer. However, we have a poor understanding of which specific EphA2 state is responsible for oncogenic signaling, which is characterized by phosphorylation of cytoplasmic serine residues. To address this question, we have developed SiMPull-POP, a new single-molecule method for accurate quantification of the self-assembly of membrane proteins. SiMPull-POP uses the amphipathic DIBMA co-polymer to isolate native-like nanodiscs from human cells. The oligomeric state of membrane proteins in the resulting DIBMALPs is determined by single molecule photobleaching. We applied SiMPull-POP to GFP-tagged EphA2. Our experiments revealed that a reduction of plasma membrane cholesterol had a strong effect on EphA2 self assembly. Indeed, low cholesterol caused a similar effect than an EphA2 ligand. These results indicate that cholesterol inhibits EphA2 assembly. Phosphorylation studies in different cells lines revealed that low cholesterol increased phospho-serine levels, the signature of oncogenic signaling. Investigation of the mechanism that cholesterol uses to inhibit the assembly and activity of EphA2 failed to reveal binding to the transmembrane domain of EphA2. Instead, our data indicate an in trans effect, where EphA2 serines are phosphorylated by protein kinase A downstream of beta-adrenergic receptor activity, which cholesterol also inhibits. Our investigation not only enriches our understanding on how EphA2 works, but it also indicates that plasma membrane cholesterol acts as a molecular safety mechanism that prevents uncontrolled self-assembly and oncogenic activation of EphA2.

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