The amino-terminal domain of g-protein-coupled receptor kinase 2 is a regulatory gβγ binding site
The amino-terminal domain of g-protein-coupled receptor kinase 2 is a regulatory gβγ binding site
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DOI：
10.1074/jbc.m204795200
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发表时间：
2003-03-07
影响因子：
4.8
通讯作者：
Quitterer, U
中科院分区：
文献类型：
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作者：
Eichmann, T;Lorenz, K;Quitterer, U
g蛋白偶联受体激酶2 （GRK2）被游离的β - γ亚基激活。GRK2的β - γ结合位点位于羧基末端pleckstrin同源结构域。GRK2的这个伽马结合位点也通过隔离游离的伽马亚基来调节伽马刺激的信号传导。我们在这里报道，截断GRK2的羧基端gbetagama结合位点并没有取消GRK2的gbetagama调节活性，这是通过抑制gbetagama刺激的细胞中肌醇磷酸增加来确定的。这一发现表明在GRK2中存在第二个gbetagama结合位点。事实上，GRK2的氨基端（GRK2(1-185)）抑制细胞中gpy刺激的肌醇磷酸信号，纯化的GRK2（1-185）抑制gbetaggamma刺激的视紫红质磷酸化，并且GRK2（1-185）直接结合纯化的gbetaggamma亚基。gbetaggamma的氨基末端调控位点与GRK-2的RGS结构域不重叠，因为RGS结构域截断的GRK2（1-53）抑制gbetaggamma介导的信号传导，其效力和功效与GRK2相似（1-185）。除了Gbetagamma调节活性外，GRK2的氨基端Gbetagamma结合位点影响GRK2的激酶活性，因为抗体特异性地与GRK2的氨基端交叉反应抑制GRK2依赖性视紫红质磷酸化。纯化的Gbetagamma亚基释放了抗体介导的抑制，这强烈表明Gbetagamma结合GRK2的氨基端增强了对视紫红质的激酶活性。因此，GRK2的氨基末端结构域是一个以前未被识别的gbetaggamma结合位点，它调节GRK2介导的受体磷酸化并抑制gbetaggamma刺激的信号传导。
G-protein-coupled receptor kinase 2 (GRK2) is activated by free Gbetagamma subunits. A Gbetagamma binding site of GRK2 is localized in the carboxyl-terminal pleckstrin homology domain. This Gbetagamma binding site of GRK2 also regulates Gbetagamma-stimulated signaling by sequestering free Gbetagamma subunits. We report here that truncation of the carboxyl-terminal Gbetagamma binding site of GRK2 did not abolish the Gbetagamma regulatory activity of GRK2 as determined by the inhibition of a Gbetagamma-stimulated increase in inositol phosphates in cells. This finding suggested the presence of a second Gbetagamma binding site in GRK2. And indeed, the amino terminus of GRK2 (GRK2(1-185)) inhibited a Gpy-stimulated inositol phosphate signal in cells, purified GRK2(1-185) suppressed the Gbetagamma-stimulated phosphorylation of rhodopsin, and GRK2(1-185) bound directly to purified Gbetagamma subunits. The amino-terminal Gbetagamma regulatory site does not overlap with the RGS domain of GRK-2 because GRK2(1-53) with truncated RGS domain inhibited Gbetagamma-mediated signaling with similar potency and efficacy as did GRK2(1-185). In addition to the Gbetagamma regulatory activity, the amino-terminal Gbetagamma binding site of GRK2 affects the kinase activity of GRK2 because antibodies specifically cross-reacting with the amino terminus of GRK2 suppressed the GRK2-dependent phosphorylation of rhodopsin. The antibody-mediated inhibition was released by purified Gbetagamma subunits, strongly suggesting that Gbetagamma binding to the amino terminus of GRK2 enhances the kinase activity toward rhodopsin. Thus, the amino-terminal domain of GRK2 is a previously unrecognized Gbetagamma binding site that regulates GRK2-mediated receptor phosphorylation and inhibits Gbetagamma-stimulated signaling.