Role of the Amino Terminus of G Protein-Coupled Receptor Kinase 2 in Receptor Phosphorylation
Role of the Amino Terminus of G Protein-Coupled Receptor Kinase 2 in Receptor Phosphorylation
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DOI：
10.1021/bi900408g
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发表时间：
2009-08-04
期刊：
影响因子：
2.9
通讯作者：
Benovic, Jeffrey L.
中科院分区：
文献类型：
--
作者：
Pao, Christina S.;Barker, Brearnn L.;Benovic, Jeffrey L.
G蛋白偶联受体激酶（GRKs）特异性磷酸化活化的G蛋白偶联受体。虽然已经确定了几种GRK的X射线晶体结构，但GRK与GPCR相互作用的机制目前尚不清楚。为了进一步表征GRK 2氨基末端在受体相互作用和磷酸化中的作用，我们在GRK 2的前10个氨基酸内产生了一系列点突变，并测试了它们磷酸化受体和非受体底物的能力。尽管所有突变体在受体磷酸化方面都表现出一定程度的损伤，但其中三个突变体D3 K、L4 A和D10 A受到的影响最严重。使用β 2-肾上腺素能受体和视紫红质作为受体底物，微管蛋白作为非受体底物，我们证明突变体对受体的激酶活性严重降低，而它们完全保留了磷酸化微管蛋白的能力。此外，氨基末端突变体能够结合到受体，但与野生型GRK 2相反，不被受体结合激活。含有GRK 2残基1-14的合成肽作为GRK 2受体磷酸化的非竞争性抑制剂，而来自GRK 5的可比肽对GRK 2活性没有影响。二级结构预测和圆二色性表明GRK 2氨基末端肽形成两亲性α-螺旋。两者合计，我们提出了一种机制，GRK 2的极端氨基末端形成分子内相互作用，选择性地增强对受体底物的激酶的催化活性。
G protein-coupled receptor kinases (GRKs) specifically phosphorylate activated G protein-coupled receptors. While the X-ray crystal structures of several GRKs have been determined, the mechanism of interaction of GRK with GPCRs is currently unknown. To further characterize the role of the GRK2 amino terminus in receptor interaction and phosphorylation, we generated a series of point mutations within the first 10 amino acids of GRK2 and tested their ability to phosphorylate receptor and nonreceptor substrates. Although all mutants exhibited some impairment in receptor phosphorylation, three of the mutants, D3K, L4A, and D10A, were the most severely affected, Using the beta(2)-adrenergic receptor and rhodopsin as receptor substrates and tubulin as a nonreceptor substrate, we demonstrated that the kinase activity toward the receptors was severely decreased in the mutants, while they fully retained their ability to phosphorylate tubulin. Moreover, the amino-terminal mutants were able to bind to the receptor but, in contrast to wild-type GRK2, were not activated by receptor binding. A synthetic peptide containing residues 1-14 of GRK2 served as a noncompetitive inhibitor of receptor phosphorylation by GRK2, while a comparable peptide from GRK5 had no effect on GRK2 activity. Secondary structure prediction and circular dichroism suggest that the GRK2 amino-terminal peptide forms an amphipathic alpha-helix. Taken together, we propose a mechanism whereby the extreme amino terminus of GRK2 forms an intramolecular interaction that selectively enhances the catalytic activity of the kinase toward receptor substrates.