A canonical EF-loop directs Ca(2+) -sensitivity in phospholipase C-η2

J Cell Biochem. 2014 Mar;115(3):557-65. doi: 10.1002/jcb.24690.

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.

Keywords: CALCIUM; CELL SIGNALING; COMPARATIVE MODELING; EF-HAND; PHOSPHOLIPASE C; SIGNAL TRANSDUCTION.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • COS Cells
  • Calcium / chemistry
  • Calcium / metabolism*
  • Calmodulin / chemistry
  • Chlorocebus aethiops
  • EF Hand Motifs / genetics
  • Humans
  • Inositol / pharmacology
  • Models, Molecular*
  • Mutation / genetics
  • Phosphoinositide Phospholipase C / chemistry*
  • Phosphoinositide Phospholipase C / metabolism
  • Protein Conformation*
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Signal Transduction
  • Structure-Activity Relationship

Substances

  • Calmodulin
  • Inositol
  • PLCH1 protein, human
  • Phosphoinositide Phospholipase C
  • Calcium