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Muscarinic agonist-induced recruitment of plasma membrane Ca2+ ATPase (PMCA) to the membrane involves the PSD95/Dlg/ZO-1 (PDZ) scaffold Na+ H+ exchanger regulatory factor 2 (NHERF-2)

W.A. Kruger,1 G.R. Monteith2 and P. Poronnik,1 1School of Biomedical Science and 2School of Pharmacy, University of Queensland, St Lucia, Qld 4072, Australia.

Despite the ubiquitous distribution of PMCA little is known about the protein-protein interactions that regulate its activity. The neuronal isoform of PMCA(2b) is known to interact with NHERF-2. The current study investigated the molecular basis and physiological role of the PMCA/NHERF-2 interaction during muscarinic-induced Ca2+ signalling in epithelial cells. HT-29 epithelial cells expressed both PMCA1 and 4 as shown by RT-PCR and Western blotting. GST/MBP-pulldowns demonstrated that PMCA bound NHERF-2 in HT29 cell lysates and mutation analysis confirmed that the interaction was mediated by the C-terminal PDZ binding motif of PMCA. Co-immunoprecipitations confirmed that PMCA and NHERF-2 were normally associated in HT29 cells. Cell surface biotinylations were used to show that cell surface PMCA increased in response to carbachol (CCh) to 154±12%; (p<0.01; n=4) of control levels within 60s. Interestingly, the recruitment of NHERF-2 to the membrane preceded PMCA, with membrane associated NHERF-2 increasing within 30s to 145.3±4.9% of control (p<0.01, n=4). In contrast, silencing NHERF-2 abolished the CCh evoked trafficking of PMCA, and reduced the levels of PMCA at the membrane to 54±5% (p<0.01; n=3) of control. Treatment with CCh also resulted in co-localisation of PMCA/NHERF-2 at the plasma membrane as determined by confocal microscopy. These data show 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.