Calmodulin potentiates Gβγ activation of phospholipase C-β3

Phospholipase C-β (PLC-β) isozymes (EC 3.1.4.11) hydrolyze the membrane phospholipid phosphatidylinositol-4,5-bisphosphate to generate intracellular second messenger signaling molecules inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) in response to receptor activation and other cellular stimuli. PLCβ1 and PLCβ3 isozymes were previously demonstrated to bind the calcium-sensitive molecule calmodulin [McCullar JS, Larsen SA, Millimaki RA, Filtz TM. Calmodulin is a phospholipase C-{beta} interacting protein. J Biol Chem 2003;278(36):33708-13]. We have now shown through fluorescence anisotropy that calmodulin/PLCβ3 affinities increase with increasing calcium in a physiologically relevant concentration range. The bimolecular affinity constants for calmodulin interaction with PLCβ1 or PLCβ3 were estimated as 260 and 200 nM, respectively, from fluorescence anisotropy data. There was no effect of calmodulin on basal or Gαq-stimulated catalytic activity for either isozyme. However, the interaction between calmodulin and PLCβ3 leads to potentiation of activation by the G-protein βγ dimer in an in vitro assay. 1321N1 cells treated with calmodulin inhibitors concurrent with and post-stimulation of muscarinic receptors significantly reduced [3H]PIP hydrolysis. Together these data are suggestive of cooperative role for calmodulin in the G-protein βγ dimer-stimulated activity of PLCβ3.