Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)
Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)
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DOI：
10.1074/jbc.m114.634329
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发表时间：
2015-04-03
影响因子：
4.8
通讯作者：
Hepler, John R.
中科院分区：
文献类型：
--
作者：
Brown, Nicole E.;Goswami, Devrishi;Hepler, John R.
RGS 14含有不同的结合位点，用于活性（GTP结合）和非活性（GDP结合）形式的G α亚基。G蛋白信号传导（RGS）结构域的N-末端调节物结合活性G α（i/o）-GTP，而C-末端G蛋白调节（GPR）基序结合非活性G α（i1/3）-GDP。RGS 14如何结合G α蛋白的不同活化状态以整合G蛋白信号传导的分子基础尚不清楚。在这里，我们探讨了GPR基序和RGS结构域之间的G蛋白结合的分子内通信，并检查RGS 14是否可以同时与两种不同形式的G α亚基功能相互作用。使用互补的细胞和生物化学方法，我们证明，RGS 14形成一个稳定的复合物与非活性G α（i1）-GDP在质膜和游离的胞质RGS 14被招募到质膜激活G α（o）-AlF 4-。生物发光共振能量转移研究表明，RGS 14在不同的激活状态下与G α结合时，在活细胞中采用不同的构象。氢/氘交换质谱法显示，RGS 14是一种非常动态的蛋白质，当无活性的G α（i1）-GDP结合GPR基序时，其经历变构构象变化。纯RGS 14与G α（o）-AlF 4-和G α（i1）-GDP的AlF 4-不敏感突变体（G42 R）形成三元复合物，如通过尺寸排阻色谱和差分氢/氘交换所观察到的。最后，预先形成的RGS 14中心点G α（i1）-GDP复合物显示出刺激G α（o）-GTP的GTP酶活性的全部能力，表明RGS 14可以在功能上同时接合两种不同形式的G α亚基。基于这些发现，我们提出了一个工作模型，RGS 14如何整合多种G蛋白信号在宿主CA 2海马神经元调节突触可塑性。
RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of G alpha subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active G alpha(i/o)-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive G alpha(i1/3)-GDP. The molecular basis for how RGS14 binds different activation states of G alpha proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of G alpha subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive G alpha(i1)-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated G alpha(o)-AlF4-. Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to G alpha in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive G alpha(i1)-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with G alpha(o)-AlF4- and an AlF4--insensitive mutant (G42R) of G alpha(i1)-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14 center dot G alpha(i1)-GDP complex exhibits full capacity to stimulate the GTPase activity of G alpha(o)-GTP, demonstrating that RGS14 can functionally engage two distinct forms of G alpha subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity.