Muscarinic-induced recruitment of plasma membrane Ca 2+-ATPase involves PSD-95/Dlg/Zo-1-mediated interactions

Abstract

Efflux of cytosolic Ca 2+ mediated by plasma membrane Ca 2+-ATPases (PMCA) plays a key role in fine tuning the magnitude and duration of Ca 2+ signaling following activation of G-protein-coupled receptors. However, the molecular mechanisms that underpin the trafficking of PMCA to the membrane during Ca 2+ signaling remain largely unexplored in native cell models. One potential mechanism for the recruitment of proteins to the plasma membrane involves PDZ interactions. In this context, we investigated the role of PMCA interactions with the Na +/H + exchanger regulatory factor 2 (NHERF-2) during muscarinicinduced Ca 2+ mobilization in the HT-29 epithelial cell line. GST pull-downs in HT-29 cell lysates showed that the PDZ2 module of NHERF-2 bound to the PDZ binding motif on the C terminus of PMCA. Co-immunoprecipitations confirmed that PMCA1b and NHERF-2 associated under normal conditions in HT-29 cells. Cell surface biotinylations revealed significant increases in membrane-associated NHERF-2 and PMCA within 60 s following muscarinic activation, accompanied by increased association of the two proteins as seen by confocal microscopy. The recruitment of NHERF-2 to the membrane preceded that of PMCA, suggesting that NHERF-2 was involved in nucleating an efflux complex at the membrane. The muscarinic-mediated translocation of PMCA was abolished when NHERF-2 was silenced, and the rate of relative Ca 2+ efflux was also reduced. These experiments also uncovered a NHERF-2-independent PMCA retrieval mechanism. Our findings describe rapid agonist-induced translocation of PMCA in a native cell model and suggest that NHERF-2 plays a key role in scaffolding and maintaining PMCA at the cell membrane. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.