Muscarinic-induced recruitment of plasma membrane Ca2+-ATPase involves PSD-95/Dlg/Zo-1-mediated interactions.
Efflux of cytosolic Ca2+ mediated by plasma membrane Ca2+-ATPases (PMCA) plays a key role in fine tuning the magnitude and duration of Ca2+ signaling following activation of G-protein-coupled receptors. However, the molecular mechanisms that underpin the trafficking of PMCA to the membrane during Ca2+ 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 muscarinic-induced Ca2+ 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 Ca2+ 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.
Kruger
Wade A
WA
School of Biomedical Sciences and School of Pharmacy, The University of Queensland, Brisbane QLD 4072, Australia.
Yun
C Chris
CC
Monteith
Gregory R
GR
Poronnik
Philip
P
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D045744:Cell Line, Tumor; D018678:Cholinergic Agents / Q000494:pharmacology*; D000074288:Discs Large Homolog 1 Protein; D000076122:Disks Large Homolog 4 Protein; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
C506952:ATP2B1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D018678:Cholinergic Agents; C513314:DLG1 protein, human; C501444:DLG4 protein, human; D000074288:Discs Large Homolog 1 Protein; D000076122:Disks Large Homolog 4 Protein; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; C093376:sodium-hydrogen exchanger regulatory factor; D053499:Plasma Membrane Calcium-Transporting ATPases; D002118:Calcium