Differential contribution of GTPase activation and effector antagonism to the inhibitory effect of RGS proteins on Gq-mediated signaling in vivo
Differential contribution of GTPase activation and effector antagonism to the inhibitory effect of RGS proteins on Gq-mediated signaling in vivo
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DOI：
10.1074/jbc.m309496200
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发表时间：
2004-02-06
影响因子：
4.8
通讯作者：
Mende, U
中科院分区：
文献类型：
--
作者：
Anger, T;Zhang, W;Mende, U
RGS蛋白通过作为异源三聚体G蛋白（Ga）的a亚基的GTP酶激活蛋白（GAP）而充当G蛋白信号传导的负调节剂，从而加速G蛋白失活。RGS蛋白还可以通过与下游效应物竞争Ga结合来阻断Ga介导的信号产生。关于GAP和效应物拮抗对RGS蛋白对G蛋白介导的信号传导的抑制作用的相对贡献知之甚少。通过比较RGS 2、RGS 3、RGS 5和RGS 16在GT α可能激活与不存在激活的条件下对Ga（q）介导的磷脂酶C β（PLC β）激活的抑制作用，我们证明R4 RGS亚家族成员在对GT α加速的依赖性方面存在显著差异。COS-7细胞用毒蕈碱M-3受体或组成型活性G α（q）瞬时转染，毒蕈碱M-3受体与内源性G（q）蛋白偶联并介导卡巴胆碱对PLC β的刺激作用，组成型活性G α（q）对GTP水解呈惰性并独立于受体活化而活化PLC β。在表达M-3的细胞中，所有RGS蛋白都显著减弱了卡巴胆碱的功效和效力。相比之下，G α（q）* 诱导的PLC β激活被RGS 2和RGS 3抑制，但不被RGS 5和RGS 16抑制。观察到的差异效应不是由于M-3、Ga（q）/Ga（q）*、PLC β或RGS表达的变化，如受体结合试验和蛋白质印迹所示。我们的结论是，密切相关的R4 RGS家族成员在其作用机制不同。RGS 5和RGS 16似乎依赖于G蛋白失活，而GAP非依赖性机制（如效应拮抗作用）足以介导RGS 2和RGS 3的抑制作用。
RGS proteins act as negative regulators of G protein signaling by serving as GTPase-activating proteins (GAP) for a subunits of heterotrimeric G proteins (Galpha), thereby accelerating G protein inactivation. RGS proteins can also block Galpha-mediated signal production by competing with downstream effectors for Ga binding. Little is known about the relative contribution of GAP and effector antagonism to the inhibitory effect of RGS proteins on G protein-mediated signaling. By comparing the inhibitory effect of RGS2, RGS3, RGS5, and RGS16 on Galpha(q)mediated phospholipase Cbeta (PLCbeta) activation under conditions where GTPase activation is possible versus nonexistent, we demonstrate that members of the R4 RGS subfamily differ significantly in their dependence on GTPase acceleration. COS-7 cells were transiently transfected with either muscarinic M-3 receptors, which couple to endogenous G(q) protein and mediate a stimulatory effect of carbachol on PLCbeta, or constitutively active Galpha(q), which is inert to GTP hydrolysis and activates PLCbeta independent of receptor activation. In M-3-expressing cells, all of the RGS proteins significantly blunted the efficacy and potency of carbachol. In contrast, Galpha(q)*-induced PLCbeta activation was inhibited by RGS2 and RGS3 but not RGS5 and RGS16. The observed differential effects were not due to changes in M-3, Galpha(q)/ Galpha(q)*, PLCbeta, or RGS expression, as shown by receptor binding assays and Western blots. We conclude that closely related R4 RGS family members differ in their mechanism of action. RGS5 and RGS16 appear to depend on G protein inactivation, whereas GAP-independent mechanisms (such as effector antagonism) are sufficient to mediate the inhibitory effect of RGS2 and RGS3.