Calmodulin potentiates G beta gamma activation of phospholipase C-beta3.
Phospholipase C-beta (PLC-beta) 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. PLCbeta1 and PLCbeta3 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/PLCbeta3 affinities increase with increasing calcium in a physiologically relevant concentration range. The bimolecular affinity constants for calmodulin interaction with PLCbeta1 or PLCbeta3 were estimated as 260 and 200 nM, respectively, from fluorescence anisotropy data. There was no effect of calmodulin on basal or G alpha q-stimulated catalytic activity for either isozyme. However, the interaction between calmodulin and PLCbeta3 leads to potentiation of activation by the G-protein beta gamma 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 beta gamma dimer-stimulated activity of PLCbeta3.
McCullar
Jennifer S
JS
Molecular and Cellular Biology Graduate Program, Oregon State University, Corvallis, OR 97331, USA.
Malencik
Dean A
DA
Vogel
Walter K
WK
Crofoot
Kristi M
KM
Anderson
Sonia R
SR
Filtz
Theresa M
TM
D002147:Calmodulin / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D005454:Fluorescence Polarization; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D054799:Phospholipase C beta; D011976:Receptors, Muscarinic / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism*
D002147:Calmodulin; D007527:Isoenzymes; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins