Friday, May 9, 2025
5/9/2025
EphA1 homotypic and EphA1-EphA2 heterotypic interactions in cell regulation - Project Summary EphA1 and EphA2 are two closely related members among the 14 Eph receptor tyrosine kinases; they critically regulate diverse physiological and pathological processes. While A2 has been extensively investigated, little is known about A1 structure, signaling and function. Using a time-resolved fluorescence spectroscopy known as Pulsed Interleaved Excitation-Fluorescence Cross-Correlation Spectroscopy (PIE-FCCS), we discovered a strong hetero-interaction between A1 and A2 in the plasma membrane of live cells. Furthermore, functional studies show that A2 dictates the function of A1 within the heterotypic complex. When present alone, A1 and A2 have distinct homotypic molecular organizations. Unliganded A2 alone self-assembles into homotypic multimers through three interfaces and undergoes rapid clustering upon ligand binding. Surprisingly, A1 instead forms dimers that are refractory to ligand-induced clustering, unveiling unexpected and exciting differences between the two related receptors. In previous reports, we established that A2 has dual functions, depending on its interaction with ligands. 1) Ligand-induced canonical tumor-suppressive signaling, characterized by the catalytic activation of the A2 tyrosine kinase, that suppresses Ras/ERK and PI3K/Akt pathways and inhibits cell migration and growth. 2) Ligand-independent non-canonical oncogenic signaling through serine 897 phosphorylation (pS897) that promotes cell migration and growth. Consistent with biophysical results from PIE-FCCS, cellular and biochemical studies show dramatic differences in A1 and A2 signaling. A1 by itself is unresponsive to ligand and does not mediate canonical signaling. A1 is phosphorylated on S906 (pS906), the residue corresponding to A2 S897, suggesting noncanonical signaling like pS897-A2. However, noncanonical signaling by A1 and A2 is differentially regulated by ligand. Importantly, we discovered that hetero-interaction with A2 transforms A1 function, whereby A1 acquires ligand responsiveness, becoming catalytically active in sync with A2. The A1/A2 hetero-interactions are physiologically and pathologically relevant as they are widely co-expressed in many epithelial tissues, including the liver. Genetic studies in mice revealed that A1 and A2 exert opposite roles in controlling tumor susceptibility: Deletion of A1 markedly suppressed hepatocarcinogenesis, whereas knockout of A2 strongly promoted it. The overarching goal of this proposal is to elucidate the molecular details of A1 homo- dimerization and A1-A2 hetero-interaction, and to delineate their roles in A1 and A2 signaling and functions. In Aim 1 we will determine the molecular basis of EphA1-EphA2 hetero-interaction. Aim 2 will characterize the interfaces that mediates EphA1 homo-dimerization. The importance of the EphA1-EphA2 hetero-interaction and EphA1 homo-dimerization in regulating cell signaling and function, including migration and invasion both in vitro and in vivo, will be investigated in Aim 3. Our findings will add novel knowledge to the understanding of EphA1 and EphA2 in human cancers including hepatocellular carcinoma and provide potential new therapeutic targets.
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