Pronin A N, Carman C V, Benovic J L
Departments of Biochemistry and Molecular Pharmacology and Microbiology and Immunology, Kimmel Cancer Cancer, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Biol Chem. 1998 Nov 20;273(47):31510-8. doi: 10.1074/jbc.273.47.31510.
Many G protein-coupled receptors are phosphorylated and regulated by a distinct family of G protein-coupled receptor kinases (GRKs) that specifically target the activated form of the receptor. Recent studies have revealed that the GRKs are also subject to post-translational regulation. For example, GRK5 activity is strongly inhibited by protein kinase C phosphorylation and by Ca2+-calmodulin binding. Ca2+-calmodulin binding also promotes GRK5 autophosphorylation, which further contributes to kinase inhibition. In this study we identify two important structural domains in GRK5, a phospholipid binding domain (residues 552-562) and an autoinhibitory domain (residues 563-590), that significantly contribute to GRK5 localization and function. We demonstrate that the C-terminal region of GRK5 (residues 563-590) contains residues autophosphorylated in the presence of calmodulin as well as the residues phosphorylated by protein kinase C. Deletion of this domain increases the apparent affinity of GRK5 for receptor substrates 3-4-fold but has no effect on nonreceptor substrates. These findings define residues 563-590 of GRK5 as an autoinhibitory domain with efficacy that is regulated by phosphorylation. Another C-terminal domain in GRK5 that appears to be functionally important is found between residues 552 and 562. Deletion of this region significantly inhibits kinase phosphorylation of membrane-bound receptor substrates but has no effect on soluble substrates. Additional studies reveal that this domain is critical for GRK5 interaction with phospholipids and for the intracellular localization of the kinase. Interestingly, similar regions in GRK4 and GRK6 appear to be palmitoylated (and involved in membrane interaction), suggesting evolutionary conservation of the function of this domain.
许多G蛋白偶联受体可被一类独特的G蛋白偶联受体激酶（GRK）磷酸化并调节，这类激酶特异性作用于受体的活化形式。最近的研究表明，GRK自身也受到翻译后修饰的调控。例如，GRK5的活性受到蛋白激酶C磷酸化以及Ca2+-钙调蛋白结合的强烈抑制。Ca2+-钙调蛋白结合还可促进GRK5的自身磷酸化，这进一步导致激酶活性的抑制。在本研究中，我们确定了GRK5中的两个重要结构域，一个磷脂结合结构域（第552 - 562位氨基酸残基）和一个自身抑制结构域（第563 - 590位氨基酸残基），它们对GRK5的定位和功能有显著贡献。我们证明，GRK5的C末端区域（第563 - 590位氨基酸残基）包含在钙调蛋白存在下发生自身磷酸化的残基以及被蛋白激酶C磷酸化的残基。缺失该结构域可使GRK5对受体底物的表观亲和力增加3 - 4倍，但对非受体底物没有影响。这些发现将GRK5的第563 - 590位氨基酸残基定义为一个自身抑制结构域，其活性受磷酸化调节。GRK5中另一个在功能上似乎很重要的C末端结构域位于第552和562位氨基酸残基之间。缺失该区域可显著抑制膜结合受体底物的激酶磷酸化，但对可溶性底物没有影响。进一步的研究表明，该结构域对于GRK5与磷脂的相互作用以及激酶的细胞内定位至关重要。有趣的是，GRK4和GRK6中的类似区域似乎发生了棕榈酰化修饰（并参与膜相互作用），这表明该结构域功能在进化上具有保守性。
