期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 275, 期 14, 页码 10443-10452出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.275.14.10443
关键词
-
资金
- NCI NIH HHS [5-T32-CA09662] Funding Source: Medline
- NIGMS NIH HHS [GM44944] Funding Source: Medline
Agonist-dependent regulation of G protein-coupled receptors is dependent on their phosphorylation by G protein-coupled receptor kinases (GRKs). GRK2 and GRK3 are selectively regulated in vitro by free G-beta-gamma subunits and negatively charged membrane phospholipids through their pleckstrin homology (PH) domains. However, the molecular binding determinants and physiological role for these ligands remain unclear. To address these issues, we generated an array of site-directed mutants within the GRK2 PH domain and characterized their interaction with G-beta-gamma and phospholipids in vitro. Mutation of several residues in the loop 1 region of the PH domain, including Lys-567, Trp-576, Arg-578, and Arg-579, resulted in a loss of receptor phosphorylation, likely via disruption of phospholipid binding, that was reversed by G-beta-gamma. Alternatively, mutation of residues distral to the C-terminal amphipathic alpha-helix, including Lys-663, Lys-665, Lys-667, and Arg-669, resulted in decreased responsiveness to G-beta-gamma. Interestingly, mutation of Arg-587 in beta-sheet 3, a region not previously thought to interact with G-beta-gamma, resulted in a specific and profound loss of G-beta-gamma responsiveness. To further characterize these effects, two mutants (GRK2(K567E/R578E) and GRK2(R587Q)) were expressed in Sf9 cells and purified. Analysis of these mutants revealed that GRK2(K567E/R578E) was refractory to stimulation by negatively charged phospholipids but bound G-beta-gamma similar to wild type GRK2. In contrast, GRK2(R587Q) was stimulated by acidic phospholipids but failed to bind G-beta-gamma. In order to examine the role of phospholipid and G-beta-gamma interaction in cells, wild-type and mutant GRK2s were expressed with a beta-2-adrenergic receptor (beta-2AR) mutant that is responsive to GRK2 phosphorylation (beta-2AR(Y326A)). In these cells, GRK2 (K567E/R578E) and GRK2(R587Q) were largely defective in promoting agonist-dependent phosphorylation and internalization of beta-2AR(Y326A). Similarly, wild-type GRK2 but not GRK2(K567E/R578E) or GRK2(R587Q) promoted morphine dependent phosphorylation of the mu-opioid receptor in cells. Thus, we have (i) identified several specific GRK2 binding determinants for G-beta-gamma and phospholipids, and (ii) demonstrated the G-beta-gamma binding is the limiting step for GRK2-dependent receptor phosphorylation in cells.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据