Carman C V, Lisanti M P, Benovic J L
Departments of Biochemistry and Molecular Pharmacology and Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Biol Chem. 1999 Mar 26;274(13):8858-64. doi: 10.1074/jbc.274.13.8858.
G protein-coupled receptor kinases (GRKs) have been principally characterized by their ability to phosphorylate and desensitize G protein-coupled receptors. However, recent studies suggest that GRKs may have more diverse protein/protein interactions in cells. Based on the identification of a consensus caveolin binding motif within the pleckstrin homology domain of GRK2, we tested the direct binding of purified full-length GRK2 to various glutathione S-transferase-caveolin-1 fusion proteins, and we discovered a specific interaction of GRK2 with the caveolin scaffolding domain. Interestingly, analysis of GRK1 and GRK5, which lack a pleckstrin homology domain, revealed in vitro binding properties similar to those of GRK2. Maltose-binding protein caveolin and glutathione S-transferase-GRK fusion proteins were used to map overlapping regions in the N termini of both GRK2 and GRK5 that appear to mediate conserved GRK/caveolin interactions. In vivo association of GRK2 and caveolin was suggested by co-fractionation of GRK2 with caveolin in A431 and NIH-3T3 cells and was further supported by co-immunoprecipitation of GRK2 and caveolin in COS-1 cells. Functional significance for the GRK/caveolin interaction was demonstrated by the potent inhibition of GRK-mediated phosphorylation of both receptor and peptide substrates by caveolin-1 and -3 scaffolding domain peptides. These data reveal a novel mode for the regulation of GRKs that is likely to play an important role in their cellular function.
G蛋白偶联受体激酶（GRKs）主要因其磷酸化G蛋白偶联受体并使其脱敏的能力而被表征。然而，最近的研究表明，GRKs在细胞中可能具有更多样化的蛋白质/蛋白质相互作用。基于在GRK2的普列克底物同源结构域内鉴定出一个共有小窝蛋白结合基序，我们测试了纯化的全长GRK2与各种谷胱甘肽S-转移酶-小窝蛋白-1融合蛋白的直接结合，并且我们发现GRK2与小窝蛋白支架结构域存在特异性相互作用。有趣的是，对缺乏普列克底物同源结构域的GRK1和GRK5的分析显示，它们在体外的结合特性与GRK2相似。麦芽糖结合蛋白小窝蛋白和谷胱甘肽S-转移酶-GRK融合蛋白被用于绘制GRK2和GRK5 N端的重叠区域，这些区域似乎介导了保守的GRK/小窝蛋白相互作用。GRK2和小窝蛋白在体内的关联通过GRK2与小窝蛋白在A431和NIH-3T3细胞中的共分级分离得到提示，并在COS-1细胞中GRK2和小窝蛋白的共免疫沉淀中得到进一步支持。小窝蛋白-1和-3支架结构域肽对GRK介导的受体和肽底物磷酸化的有效抑制证明了GRK/小窝蛋白相互作用的功能意义。这些数据揭示了一种GRKs的新型调节模式，这可能在其细胞功能中发挥重要作用。
