PhD Scientific Days 2025

Molecular Medicine IV.

ARF6 and SNX27 Coordinate Endosomal Recycling of the Plasma Membrane Calcium Pump PMCA4b

Name of the presenter

Nagy Réka

Institute/workplace of the presenter

SE Department of Transfusion Medicine

Authors

Réka Nagy¹, Sarolta Tóth¹, Gergő Gógl², Boglárka Zámbó², Ágnes Enyedi¹

1: Transfusion Medicine, Semmelweis University, Budapest, Hungary
2: Department of Integrated Structural Biology, IGBMC, Illkirch, France

Text of the abstract

Introduction: The plasma membrane Ca²⁺ ATPase (PMCA) maintains low cytosolic Ca²⁺ levels by transporting Ca²⁺ out of the cell. One of the major PMCA isoforms of non-excitable cells is PMCA4b, which regulates key processes such as cell motility and polarization by shaping intracellular calcium dynamics. Prior studies suggest that PMCA4b may act as a metastasis suppressor in BRAF mutant melanoma cells due to its impact on migration and metastatic potential.
Aims: This study aimed to investigate the endocytic trafficking and recycling of PMCA4b, focusing on its interactions with regulatory proteins that may influence its localization and function in cell motility and polarity.
Methods: We used HEK293 and MCF-7 cell lines stably expressing GFP-PMCA4b to examine its intracellular trafficking. Protein localization and interactions were investigated using immunofluorescence staining and colocalization analysis. The impact of ARF6 inhibition was assessed using its specific inhibitor NAV2729. Additionally, full-length and PDZ domain-deleted SNX27 constructs were overexpressed to evaluate their interaction with PMCA4b.
Results: PMCA4b overexpression led to increased expression of its subunit CD147/basigin in both cell lines, with strong colocalization at the plasma membrane. Inhibition of ARF6 with its specific inhibitor NAV2729 enhanced PMCA4b-CD147 accumulation at cell-cell contact sites and/or intracellular vesicles depending on cell types, suggesting that ARF6 regulates endocytic recycling of PMCA4b. The PDZ-domain containing SNX27, identified as a novel PMCA4b interactor, showed colocalization with PMCA4b at membrane protrusions in HEK293 cells and at the leading edge of migrating MCF-7 cells. Strong colocalization was observed with full-length SNX27, but not with the PDZ domain-lacking variant, indicating a PDZ-specific interaction. Immunostaining revealed that SNX27 may function as an adaptor linking PMCA4b to ARF6-positive endosomes for recycling to the plasma membrane.
Conclusion: Our findings uncover a novel mechanism for PMCA4b recycling via an ARF6-mediated pathway coordinated by SNX27. This regulatory pathway may contribute to the role of PMCA4b in regulating cell polarization, cell motility and potentially in suppressing metastatic behavior.
Funding: This work was supported by grants TKP2021-EGA-24 and 2024-1.2.3-HU-RIZONT-2024-00003.