Lodowski David T, Barnhill Jennifer F, Pyskadlo Robyn M, Ghirlando Rodolfo, Sterne-Marr Rachel, Tesmer John J G
Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 78712-0165, USA.
Biochemistry. 2005 May 10;44(18):6958-70. doi: 10.1021/bi050119q.
In response to extracellular signals, G protein-coupled receptors (GPCRs) catalyze guanine nucleotide exchange on Galpha subunits, enabling both activated Galpha and Gbetagamma subunits to target downstream effector enzymes. One target of Gbetagamma 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 Gbetagamma. The crystal structure of the GRK2-Gbetagamma complex revealed that the domains of GRK2 are intimately associated and left open the possibility for allosteric regulation by Gbetagamma. In this paper, we report the 4.5 A structure of GRK2, which shows that the binding of Gbetagamma 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 Gbetagamma, but not a soluble mutant of Gbetagamma, protects GRK2 from clostripain digestion at a site within its kinase domain that is 80 A away from the Gbetagamma binding site. Equilibrium ultracentrifugation experiments indicate that neither abnormally large detergent micelles nor protein oligomerization can account for the observed protection. The Gbetagamma-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.
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，使活化的Gα亚基和Gβγ亚基都能作用于下游效应酶。Gβγ的一个作用靶点是G蛋白偶联受体激酶2（GRK2），该酶通过磷酸化活化的GPCRs引发同源脱敏。GRK2由三个不同的结构域组成：一个RGS同源（RH）结构域、一个蛋白激酶结构域和一个普列克底物蛋白同源（PH）结构域，它通过PH结构域与Gβγ结合。GRK2 - Gβγ复合物的晶体结构表明，GRK2的结构域紧密相连，并为Gβγ的变构调节提供了可能性。在本文中，我们报道了GRK2的4.5 Å结构，该结构表明，Gβγ的结合不会在GRK2中诱导大的结构域重排，尽管RH和PH结构域相对于激酶结构域有明显的小角度旋转。GRK2较大结构域界面内的残基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很重要，并且在GRK2激活后保持完整。香叶基香叶基化的Gβγ，而不是Gβγ的可溶性突变体，能保护GRK2在其激酶结构域内一个距Gβγ结合位点80 Å的位点免受梭菌蛋白酶消化。平衡超速离心实验表明，异常大的去污剂胶束和蛋白质寡聚化都不能解释观察到的保护作用。因此，Gβγ介导的GRK2与CHAPS胶束或脂质双层的结合似乎使激酶结构域刚性化，可能是通过促进RH和激酶结构域之间的稳定接触。
