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

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Signaling

Background:

• Phospholipase C-η (PLCη) enzymes hydrolyze phosphatidylinositol 4,5-bisphosphate, playing a role in intracellular signaling.
• PLCη2 exhibits calcium (Ca2+) sensitivity, suggesting a role in amplifying transient Ca2+ signals.
• PLCη enzymes feature an EF-hand domain with a canonical EF-loop 1 and a non-canonical EF-loop 2.

Purpose of the Study:

• To investigate the role of the EF-hand domain in PLCη2's Ca2+ sensing capabilities.
• To determine which EF-loop within the EF-hand domain is responsible for Ca2+ binding and signal transduction.
• To elucidate the mechanism by which PLCη2 responds to changes in intracellular Ca2+ levels.

Main Methods:

• 2D-[(1)H,(1)H] TOCSY NMR was used to examine Ca2+ binding to synthetic peptides of PLCη2 EF-loops and calmodulin.
• Molecular modeling was employed to construct a model of the PLCη2 EF-hand domain based on calmodulin structure.
• Inositol phosphate accumulation assays were performed in COS7 cells expressing wild-type and mutant PLCη2 proteins to measure enzyme activity and Ca2+ sensitivity.

Main Results:

• Both PLCη2 EF-loop peptides bound Ca2+ similarly to calmodulin EF-loop 1, particularly at the N-terminus.
• Wild-type PLCη2 activity increased 4- to 7-fold with monensin-induced intracellular Ca2+ elevation and showed a ~4-fold increase at 1 µM free Ca2+.
• A D256A mutation in EF-loop 1 significantly reduced Ca2+ sensitivity by ~10-fold and abolished monensin response, confirming EF-loop 1's role in Ca2+ sensing.
• Mutations in EF-loop 2 (D292A, H296A, Q297A, E304A) did not significantly affect monensin response or Ca2+ sensitivity, indicating EF-loop 2 is not involved in Ca2+ sensing.

Conclusions:

• The EF-hand domain of PLCη2 is critical for its ability to sense intracellular Ca2+ levels.
• EF-loop 1 of the PLCη2 EF-hand domain is the primary Ca2+-binding site responsible for mediating Ca2+ sensitivity.
• The non-canonical EF-loop 2 does not play a significant role in the Ca2+-sensing mechanism of PLCη2.