Phospholipase C-ɳ(PLCɳ) enzymes are a class of phosphatidylinositol 4,5-bisphosphatehydrolyzing enzymes involved in intracellular signaling. PLCɳ2 can sense Ca 2+ (stimulated bỹ 1 µM free Ca 2+ ) suggesting that it can amplify transient Ca 2+ signals. PLCɳ enzymes possess an EF-hand domain composed of two EF-loops; a canonical 12-residue loop (EF-loop 1) and a non-canonical 13-residue loop (EF-loop 2). Ca 2+ -binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCɳ2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[ 1 H, 1 H] TOCSY NMR. Both PLCɳ2 EF-loop peptides bound Ca 2+ in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCɳ2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca 2+ -binding. To determine whether the EF-hand is responsible for Ca 2+ -sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLCɳ2 proteins. Addition of 70µM monensin (a Na + /H + antiporter that increases intracellular Ca 2+ ) induced a 4 to 7-fold increase in wild-type PLCɳ2 activity. In permeabilized cells, PLCn2 exhibited a ~4-fold increase in activity in the presence of 1 µM free Ca 2+ . The D256A (EF-loop1) mutant exhibited a ~10-fold reduction in Ca 2+ -sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca 2+ -sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A and E304A mutants. Interestingly, the monensin responses and Ca 2+ -sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca 2+ -sensing.
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