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

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Author(s)

Petra Popovics, Jin Lu, L Nadia Kamil, Kevin Morgan, Robert P Millar, Ralf Schmid, Claudia A Blindauer, Alan J. Stewart

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Abstract

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.

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Details

Original languageEnglish
Pages (from-to)557–565
Number of pages9
JournalJournal of Cellular Biochemistry
Volume115
Issue number3
Early online date14 Oct 2013
DOIs
Publication statusPublished - 1 Mar 2014

    Research areas

  • calcium, cell signaling, comparative modeling, EF-hand, phospholipase C, signal transduction

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