The role of gβγ and domain interfaces in the activation of G protein-coupled receptor kinase 2
The role of gβγ and domain interfaces in the activation of G protein-coupled receptor kinase 2
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DOI：
10.1021/bi050119q
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发表时间：
2005-05-10
期刊：
影响因子：
2.9
通讯作者：
Tesmer, JJG
中科院分区：
文献类型：
--
作者：
Lodowski, DT;Barnhill, JF;Tesmer, JJG
作为对细胞外信号的响应，G蛋白偶联受体(GPCRs)催化Gα亚基上的鸟嘌呤核苷酸交换，使激活的Gα和Gβγ亚基都能靶向下游的效应酶。Gβ-γ的一个靶点是G蛋白偶联受体激酶2(GRK2)，这是一种通过磷酸化激活的GPCRs来启动同源脱敏的酶。GRK2由三个不同的结构域组成：RGS同源(RH)结构域、蛋白激酶结构域和Pleckstrin同源(PH)结构域，它通过这个结构域与Gβγ结合。GRK2-Gβ-伽马复合体的晶体结构表明，GRK2的结构域是紧密相关的，并为G-β伽马对变构的调节提供了可能性。在本文中，我们报道了GRK2的4.5埃结构，这表明Gβ伽马的结合不会在GRK2中引起大的结构域重排，尽管RH和PH结构域相对于激活域有明显的小旋转。GRK2较大结构域界面内残基的突变通常会导致表达和活性降低，这表明这些界面对于稳定性是重要的，并且在GRK2激活时保持完整。Geranylgeranylgeranylated G beta Gamma，但不是G beta Gamma的可溶性突变体，保护GRK2在其激酶结构域内远离GβGamma结合位点80A的位置上免受杆菌痛的消化。平衡超速离心实验表明，无论是异常大的洗涤剂胶束还是蛋白质寡聚都不能解释观察到的保护作用。因此，Gβ-γ/介导的GRK2与CHAPS胶束或脂质双层的结合似乎通过促进RH和KK结构域之间的稳定接触而使KK结构域变得刚性。
In response to extracellular signals, G protein-coupled receptors (GPCRs) catalyze guanine nucleotide exchange on G alpha subunits, enabling both activated G alpha and G beta gamma subunits to target downstream effector enzymes. One target of G beta gamma is G protein-coupled receptor kinase 2 (GRK2), an enzyme that initiates homologous desensitization by phosphorylating activated GPCRs. GRK2 consists of three distinct domains: an RGS homology (RH) domain, a protein kinase domain, and a pleckstrin homology (PH) domain, through which it binds G beta gamma. The crystal structure of the GRK2-G beta gamma complex revealed that the domains of GRK2 are intimately associated and left open the possibility for allosteric regulation by G beta gamma. In this paper, we report the 4.5 angstrom structure of GRK2, which shows that the binding of G beta gamma does not induce large domain rearrangements in GRK2, although small rotations of the RH and PH domains relative to the kinase domain are evident. Mutation of residues within the larger domain interfaces of GRK2 generally leads to diminished expression and activity, suggesting that these interfaces are important for stability and remain intact upon activation of GRK2. Geranylgeranylated G beta gamma, but not a soluble mutant of G beta gamma, protects GRK2 from clostripain digestion at a site within its kinase domain that is 80 A away from the G beta gamma binding site. Equilibrium ultracentrifugation experiments indicate that neither abnormally large detergent micelles nor protein oligornerization can account for the observed protection. The G beta gamma/mediated binding of GRK2 to CHAPS micelles or lipid bilayers therefore appears to rigidify the kinase domain, perhaps by encouraging stable contacts between the RH and kinase domains.