Regulation of phospholipase C-beta isozymes by calmodulin Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/8k71nk801

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  • Phospholipase C-Jβ (PLCβ) is an important effector enzyme in G-protein-coupled signaling pathways. Activation of PLCβ by Gα and Gβy subunits has been fairly well characterized, but little is known about other protein interactions that may also regulate PLCβ function. A yeast two-hybrid screen of a mouse brain cDNA library with the amino-terminus of PLCβ yielded potential PLCβ3-interacting proteins including calmodulin (CaM). Physical interaction between CaM and PLCβ3 was supported by a positive secondary screen and the identification in silico of a CaM binding site in the amino terminus of PLCβ3. Co-precipitation of in vitro transcribed and translated amino- and carboxy-terminal PLCβ3 fragments revealed CaM binding at a putative amino-terminal binding site. Direct physical interaction of PLCβ3 or PLCβ1 isoforms with CaM was supported by pull-down of both isoenzymes with CaM-sepharose beads from 132 1N1 cell lysates. CaM inhibitors reduced muscarinic receptor stimulation of inositol phospholipid (IP) hydrolysis in 132 iN 1 astrocytoma cells consistent with a physiologic role for CaM in modulation of PLCβ activity. CaM inhibitors significantly reduced 3H-PIP hydrolysis in 132 1N1 cells under various activation conditions. There was no effect of CaM kinase II inhibitors on M1- muscarinic receptor stimulation of IP hydrolysis, consistent with a direct interaction between PLCβ isoforms and CaM. CaM-Sepharose pull-down experiments with PLCβ3 fragments, deletion and truncation mutations show the CaM binding site residing within the putative EF-hand domains of PLCβ3. Fluorescent anisotropy data was used to calculate the binding affinity between CaM and PLCβ1 or PLCβ3 as 260 nM and 320 nM, respectively. The binding interaction between PLCβ3 and CaM appears to occur under physiologically relevant Ca2 concentrations. There was no effect of CaM on basal- and Gαq-stimulated PLCβ1 or PLCβ3 activity. Instead, the interaction between CaM and PLCβ3 leads to potentiation of activation by Gβy in an in vitro hydrolysis assay. CaM does not alter the affinity of PLCβ1 or PLCβ3 for the membrane phospholipids immobilized on nitrocellulose. However, these experiments revealed a previously unappreciated binding of PLCβ3 to PIP3 and specificity for lipid side-chain composition. This work furthers our understanding of the regulation of the PLC isozymes by identifying and characterizing the novel PLCβ activity potentiating protein, CaM.
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