期刊
CELLULAR SIGNALLING
卷 21, 期 9, 页码 1379-1384出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.cellsig.2009.04.005
关键词
Phosphatidic acid; Phospholipase C-beta; G alpha(q); GPCR; Phospholipase D; Diacylglycerol kinase
类别
Phosphatidic acid (PA), generated downstream of monomeric Rho GTPases via phospholipase D (PLD) and additionally by diacylglycerol kinases (DGK), both stimulates phospholipase C-beta(1), (PLC-beta(1)) and potentiates stimulation of PLC-beta(1) activity by G alpha(q) in vitro. PA is a potential candidate for integrating signaling by monomeric and heterotrimeric G proteins to regulate signal output by G protein coupled receptors (GPCR), and we have sought to understand the mechanisms involved. We previously identified the region spanning residues 944-957, lying within the PLC-beta(1) C-terminus alpha A helix and flexible loop of the G alpha(q) binding domain, as required for stimulation of lipase activity by PA in vitro. Regulation by PA does not require residues essential for stimulation by G alpha(q) or GTPase activating activity. The present studies evaluated shorter alanine/glycine replacement mutants and finally point mutations to identify Tyr(952) and Ile(955) as key determinants for regulation by PA, assessed by both in vitro enzymatic and cell-based co-transfection assays. Replacement of Tyr(952) and Ile(955), PLC-beta(1) (Y952G/I955G), results in an 85% loss in stimulation by PA relative to WT-PLC-beta(1) in vitro. COS 7 cells co-transfected with PLC-beta(1) (Y952G/I955G) demonstrate a 10-fold increase in the EC50 for stimulation and a 60% decrease in maximum stimulation by carbachol via G alpha(q) linked m1 muscarinic receptors, relative to cells co-transfected with WT-PLC-beta(1) but otherwise similar conditions. Residues required for regulation by PA are not essential for stimulation by G protein subunits. WT-PLC-beta(1) and PLC-beta(1) (Y952G/I955G) activity is increased comparably by co-transfection with G alpha(q) and neither is markedly affected by co-transfection with G beta(1)gamma(2). Inhibiting PLD-generated PA production by 1-butanol has little effect on maximum stimulation, but shifts the EC50 for agonist stimulation of WT-PLC-beta(1) by 10-fold, producing a phenotype similar to PLC-beta(1) (Y952G/I955G) with respect to agonist potency. 1-Butanol is without effect on carbachol stimulated PLC activity in cells co-transfected with either PLC-beta(1)(Y952G/I955G) or on endogenous PLC activity, indicating that regulation by PA requires direct interaction with the PLC-beta(1) PA-binding region. These data show that endogenous PA regulates signal output by G alpha(q)-linked GPCRs in transfected cells directly through PLC-beta(1). G alpha(q) and PA may co-ordinate to regulate signaling. Regulation by PA may constitute part of a mechanism that routes receptor signaling to specific PLC isoforms. (C) 2009 Elsevier Inc. All rights reserved.
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