G-protein-coupled receptor agonists activate endogenous phospholipase Cε and phospholipase Cβ3 in a temporally distinct manner

被引:67
作者
Kelley, GG [1 ]
Kaproth-Joslin, KA
Reks, SE
Smrcka, AV
Wojcikiewicz, RJH
机构
[1] SUNY Upstate Med, Dept Med, Syracuse, NY 13210 USA
[2] SUNY Upstate Med, Dept Pharmacol, Syracuse, NY 13210 USA
[3] Univ Rochester, Sch Med & Dent, Dept Pharmacol, Rochester, NY 14642 USA
关键词
D O I
10.1074/jbc.M507681200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Phospholipase C epsilon (PLC epsilon) is one of the newest members of the phosphatidylinositol-specific phospholipase C (PLC) family. Previous studies have suggested that G-protein-coupled receptors (GPCRs) stimulate phosphoinositide (PI) hydrolysis by activating PLC beta isoforms through G(q) family G proteins and G beta gamma subunits. Using RNA interference to knock down PLC isoforms, we demonstrate that the GPCR agonists endothelin (ET-1), lysophosphatidic acid (LPA), and thrombin, acting through endogenous receptors, couple to both endogenous PLC epsilon and the PLC beta isoform, PLC beta 3, in Rat-1 fibroblasts. Examination of the temporal activation of these PLC isoforms, however, reveals agonist- and isoform-specific profiles. PLC beta 3 is activated acutely within the first minute of ET-1, LPA, or thrombin stimulation but does not contribute to sustained PI hydrolysis induced by LPA or thrombin and accounts for only part of ET-1 sustained stimulation. PLC epsilon, on the other hand, predominantly accounts for sustained PI hydrolysis. Consistent with this observation, reconstitution of PLC epsilon in knockdown cells dose-dependently increases sustained, but not acute, agonist- stimulated PI hydrolysis. Furthermore, combined knockdown of both PLC epsilon and PLC beta 3 additively inhibits PI hydrolysis, suggesting independent regulation of each isoform. Importantly, ubiquitination of inositol 1,4,5-trisphosphate receptors correlates with sustained, but not acute, activation of PLC epsilon or PLC beta 3. In conclusion, GPCR agonists ET-1, LPA, and thrombin activate endogenous PLC epsilon and PLC beta 3 in Rat-1 fibroblasts. Activation of these PLC isoforms displays agonist- specific temporal profiles; however, PLC beta 3 is predominantly involved in acute and PLC epsilon in sustained PI hydrolysis.
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收藏
页码:2639 / 2648
页数:10
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