Plasma membrane Ca2+-ATPase associates with the cytoskeleton in activated platelets through a PDZ-binding domain

The plasma membrane Ca2+-ATPase (PMCA) plays an essential role in maintaini ng low cytosolic Ca2+ in resting platelets. During platelet activation PMCA is phosphorylated transiently on tyrosine residues resulting in inhibition of the pump that enhances elevation of Ca2+ Tyrosine phosphorylation of ma ny proteins during platelet activation results in their association with th e cytoskeleton. Consequently, in the present study we asked if PMCA interac ts with the platelet cytoskeleton, We observed that very little PMCA is ass ociated with the cytoskeleton in resting platelets but that similar to 80% of total PMCA (PMCA1b + PMCA4b) is redistributed to the cytoskeleton upon a ctivation with thrombin, Tyrosine phosphorylation of PMCA during activation was not associated with the redistribution because tyrosine-phosphorylated PMCA was not translocated specifically to the cytoskeleton. Because PMCA b -splice isoforms have C-terminal PSD-95/Dlg/ZO-1 homology domain (PDZ)-bind ing domains, a C-terminal peptide was used to disrupt potential PDZ domain interactions. Activation of saponin-permeabilized platelets in the presence of the peptide led to a significant decrease of PMCA in the cytoskeleton. PMCA associated with the cytoskeleton retained Ca2+-ATPase activity. These results suggest that during activation active PMCA is recruited to the cyto skeleton by interaction with PDZ domains and that this association provides a microenvironment with a reduced Ca2+ concentration.