A canonical EF-loop directs Ca²⁺-sensitivity in phospholipase C-η2

Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing enzymes involved in intracellular signaling. PLCη2 can sense Ca²⁺ (stimulated by ~1 μM free Ca²⁺) suggesting that it can amplify transient Ca2+ signals. PLCη 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). Ca²⁺-binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCη2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[¹H,¹H] TOCSY NMR. Both PLCη2 EF-loop peptides bound Ca²⁺ in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCη2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca²⁺-binding. To determine whether the EF-hand is responsible for Ca²⁺-sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLCη2 proteins. Addition of 70 μM monensin (a Na⁺/H⁺ antiporter that increases intracellular Ca²⁺) induced a 4 to 7-fold increase in wild-type PLCη2 activity. In permeabilized cells, PLCη2 exhibited a ~4-fold increase in activity in the presence of 1 μM free Ca²⁺. The D256A (EF-loop1) mutant exhibited a ~10-fold reduction in Ca²⁺-sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca²⁺-sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A and E304A mutants. Interestingly, the monensin responses and Ca²⁺-sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca²⁺-sensing.