Structure and Function of the Hypertension Variant A486V of G Protein-coupled Receptor Kinase 4
Structure and Function of the Hypertension Variant A486V of G Protein-coupled Receptor Kinase 4
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DOI：
10.1074/jbc.m115.648907
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发表时间：
2015-08-14
影响因子：
4.8
通讯作者：
Lumb, Kevin J.
中科院分区：
文献类型：
--
作者：
Allen, Samantha J.;Parthasarathy, Gopal;Lumb, Kevin J.
G蛋白偶联受体（GPCR）激酶（GRKs）结合并磷酸化GPCR，启动GPCR脱敏和内化过程。GRK 4参与血压的调节，并且三种GRK 4多态性（R65 L、A142 V和A486 V）与高血压相关。在此，我们描述了在5 ′-腺苷酰β，γ-亚氨基二磷酸存在下结晶的人GRK 4 α A486 V的2.6埃结构。GRK 4 α的结构与其他GRK相似，尽管在RGS同源（RH）束亚结构域、底物结合位点和激酶C尾内存在轻微差异。RH束亚结构域和激酶C-末端叶形成明显的酸性表面，而激酶N-末端叶和RH末端亚结构域表面则碱性更强。在这方面，GRK 4 alpha比GRK 6更类似于GRK 2。完全有序的激酶C-尾揭示了将C-尾与激酶活性的重要决定因素（包括α B螺旋、α D螺旋和P-环）连接的相互作用。野生型GRK 4 α的自磷酸化是完全激酶活性所必需的，如在没有ATP预孵育的情况下多巴胺D-1受体的肽磷酸化的滞后所示。相反，在GRK 4 α A486 V中没有观察到这种滞后。通过质谱法进行的磷酸肽作图表明，相对于野生型GRK 4 α，GRK 4 α A486 V中许多残基的自磷酸化速率增加，包括激酶C-尾中的Ser-485。
G-protein-coupled receptor (GPCR) kinases (GRKs) bind to and phosphorylate GPCRs, initiating the process of GPCR desensitization and internalization. GRK4 is implicated in the regulation of blood pressure, and three GRK4 polymorphisms (R65L, A142V, and A486V) are associated with hypertension. Here, we describe the 2.6 angstrom structure of human GRK4 alpha.A486V crystallized in the presence of 5'-adenylyl beta,gamma-imidodiphosphate. The structure of GRK4 alpha is similar to other GRKs, although slight differences exist within the RGS homology (RH) bundle subdomain, substrate-binding site, and kinase C-tail. The RH bundle subdomain and kinase C-terminal lobe form a strikingly acidic surface, whereas the kinase N-terminal lobe and RH terminal subdomain surfaces are much more basic. In this respect, GRK4 alpha is more similar to GRK2 than GRK6. A fully ordered kinase C-tail reveals interactions linking the C-tail with important determinants of kinase activity, including the alpha B helix, alpha D helix, and the P-loop. Autophosphorylation of wild-type GRK4 alpha is required for full kinase activity, as indicated by a lag in phosphorylation of a peptide from the dopamine D-1 receptor without ATP preincubation. In contrast, this lag is not observed in GRK4 alpha A486V. Phosphopeptide mapping by mass spectrometry indicates an increased rate of autophosphorylation of a number of residues in GRK4 alpha A486V relative to wild-type GRK4 alpha, including Ser-485 in the kinase C-tail.