title: A canonical EF-loop directs Ca(2+) -sensitivity in phospholipase C-η2.
abstract: Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing enzymes involved in intracellular signaling. PLCη2 can sense Ca(2+) (stimulated by ∼1 µM free Ca(2+) ) suggesting that it can amplify transient Ca(2+) 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). Ca(2+) -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-[(1) H,(1) H] TOCSY NMR. Both PLCη2 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 PLCη2 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 PLCη2 proteins. Addition of 70 µM monensin (a Na(+) /H(+) antiporter that increases intracellular Ca(2+) ) 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 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.
authors.affiliation: School of Medicine, University of St Andrews, St Andrews, UK.
authors.forename: Petra
authors.initials: P
authors.lastname: Popovics
authors.forename: Jin
authors.initials: J
authors.lastname: Lu
authors.forename: L
authors.initials: L
authors.lastname: Nadia Kamil
authors.forename: Kevin
authors.initials: K
authors.lastname: Morgan
authors.forename: Robert P
authors.initials: RP
authors.lastname: Millar
authors.forename: Ralf
authors.initials: R
authors.lastname: Schmid
authors.forename: Claudia A
authors.initials: CA
authors.lastname: Blindauer
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Stewart
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry; D002522:Chlorocebus aethiops; D020832:EF Hand Motifs / Q000235:genetics; D006801:Humans; D007294:Inositol / Q000494:pharmacology; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D054801:Phosphoinositide Phospholipase C / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013329:Structure-Activity Relationship
chemical_list: D002147:Calmodulin; D007294:Inositol; C506060:PLCH1 protein, human; D054801:Phosphoinositide Phospholipase C; D002118:Calcium
keywords: CALCIUM; CELL SIGNALING; COMPARATIVE MODELING; EF-HAND; PHOSPHOLIPASE C; SIGNAL TRANSDUCTION