Muscarinic-induced Recruitment of Plasma Membrane Ca2+-ATPase Involves PSD-95/Dlg/Zo-1-mediated Interactions

Abstract

Efflux of cytosolic Ca2؉ 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.

Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca2+ Pump 2B Variants in Polarized Epithelial Cells

“…NHERF2-mediated recruitment of PMCA1b to the membrane has recently been shown to be a regulated and dynamic process; in HT-29 colon epithelial cells, endogenous PMCA1b was found to rapidly (within 60 s) translocate to the plasma membrane following muscarinic receptor stimulation, and this process was dependent on NHERF2 (39). NHERF2 translocation to the membrane preceded that of the PMCA, suggesting that regulation of NHERF2 trafficking could be a mechanism for the controlled deployment of the PMCA to specific membrane domains.…”

“…Of interest to this study, NHERF2 and its target, podocalyxin/ gp135, are involved in the establishment of epithelial membrane polarity by forming an early apical scaffold during polarization of MDCK cells (13). PMCA2b (as well as PMCA1b) interacts selectively with NHERF2 over NHERF1 (6,39). The selectivity of NHERFs for different targets might help separate signaling complexes in the plasma membrane and thereby assist cells in organizing different Ca 2ϩ -dependent mechanisms into spatially discrete Ca 2ϩ signaling domains.…”

Interference with Ubiquitination in CFTR Modifies Stability of Core Glycosylated and Cell Surface Pools

“…Furthermore, ZO-1 is often cited as a key interacting protein for plasma membrane components (24). In the experiment shown in Fig.…”

NHERF-2 maintains endothelial homeostasis

“…In neuronal and epithelial cells, NHERF-2 coprecipitates with and activates the plasma membrane Ca 2ϩ -ATPase (PMCA) efflux pumps, including PMCA 1b, the isoform expressed in ECs. [41][42][43] It is possible then that loss of interaction with NHERF-2 blocks activation of PMCA, thereby facilitating retention of cytosolic Ca 2ϩ (Figure 6). …”