Programme Contents

Na⁺ H⁺ exchanger regulatory factor 2 (NHERF-2) is a scaffold for the plasma membrane Ca²⁺ ATPase (PMCA)

Resting cytosolic Ca²⁺ levels are maintained at nanomolar levels by the sequestration of Ca²⁺ into intracellular stores or the extrusion of Ca²⁺ across the plasma membrane by the PMCA. Despite the ubiquitous distribution of PMCA and its pivotal role in Ca²⁺ signalling, little is known about how PMCA activity is regulated during G protein coupled receptor signalling. There are 4 isoforms of PMCA (1-4) and many splice variants of all isoforms have been identified and all PMCA-b splice variants have a consensus class 1 PSD-95/Dlg/Zo-1 (PDZ) binding motif (Strehler et al., 2001). Protein-protein interactions mediated by PDZ modules are now recognized as playing a key role in spatially constraining many ion channels and transporters into signalling complexes in membrane microdomains (Pawson et al., 1997). Previously, PMCA 2b has been reported to interact with NHERF-2 in a heterologous expression system (DeMarco et al., 2003). This study investigated whether PMCA interacts with NHERF-2 in a native epithelial cell and the physiological significance of this interaction in terms of G-protein mediated Ca²⁺ signalling via the muscarinic M3 receptor.

This study used the polarised epithelial HT29 cell line which expresses only the M3 isoform of the muscarinic receptor. RT-PCR and Western blotting were used to confirm the presence of both PMCA and NHERF-2 in these cells. Cell surface biotinylations were performed to investigate the changes in levels of PMCA at the plasma membrane following activation of M3 receptor by acetylcholine (ACh). NHERF-2 contains 3 binding domains, PDZ-1, PDZ-2 and the C-terminus. We used GST-fusions of these domains as well as full length NHERF-2 to characterise the interaction between NHERF-2 and PMCA. These interactions were validated in vivo using co-immunoprecipitation with a polyclonal NHERF-2 antibody and subsequent Western blotting with a pan-PMCA antibody. To examine the functional role of NHERF-2, endogenous protein was knocked down by transfecting siRNA plasmids. Changes in intracellular Ca²⁺ were measured using FURA-2 in a microplate assay.

RT-PCR and Western blots confirmed that HT29 cells expressed both NHERF-2 and PMCA isoform 1 and 4 (n = 3). Importantly, we found that the levels of PMCA at the plasma membrane increased by 62 ± 12% (n = 3) within 1 min of exposure to ACh and returned to control levels within 3 min. GST-pulldown experiments in HT29 cell lysates clearly showed that PMCA interacted with the second PDZ module of NHERF-2 (n=4). Co-immunoprecipitation experiments using HT29 cell lysates confirmed the interaction between NHERF-2 and PMCA occurred under in vivo conditions (n=3). Silencing of NHERF-2 reduced the levels of endogenous NHERF-2 by a 68 ± 10% (n = 3). When we examined the Ca²⁺ response to ACh in the cells where NHERF-2 had been silenced we observed that the rate of recovery from the peak Ca²⁺ transient was 50 ± 10% (n = 3; P < 0.05) faster than in control cells.

These data reveal for the first time that the increase in intracellular Ca²⁺ in response to M3 receptor activation is accompanied by a rapid increase in PMCA at the plasma membrane, presumably due to translocation from subplasmalemmal stores. The functional interaction between NHERF-2 and PMCA may underlie the changes in recovery rate of Ca²⁺ following exposure to ACh. Further studies will provide new insights into how scaffold proteins may confer specificity in terms of G-protein mediated Ca²⁺ signalling.

Demarco, S., Chicka, M. & Strehler, E. (2003) Journal of Biological Chemistry 277, 10506-11.

Pawson, T. & Scott, J. (1997) Science 278, 2075-80.

Strehler, E. & Zacharias, D. (2001) Physiological Reviews 81, 21-50.