Koch W J, Inglese J, Stone W C, Lefkowitz R J
Howard Hughes Medical Institute, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1993 Apr 15;268(11):8256-60.
The beta gamma subunits of heterotrimeric G proteins play important roles in regulating receptor-stimulated signal transduction processes. Recently appreciated among these is their role in the signaling events that lead to the phosphorylation and subsequent desensitization of muscarinic cholinergic (Haga, K., and Haga, T. (1992) J. Biol. Chem. 267, 2222-2227) and beta-adrenergic (Pitcher, J. A., Inglese, J., Higgins, J. B., Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264-1267) receptors. Beta gamma mediates the membrane targeting of the beta-adrenergic receptor kinase (beta ARK), in response to receptor activation, through a specific beta ARK-beta gamma interaction. This process utilizes the membrane-anchoring properties of the isoprenylated gamma subunit of beta gamma. In the present study, we have employed three distinct approaches to identify the region within the carboxyl terminus of beta ARK which binds beta gamma and thereby results in membrane translocation. We studied the ability of beta gamma to enhance the enzymatic activity of a series of truncated mutants of bovine beta ARK1, the ability of glutathione S-transferase fusion proteins containing various lengths of the carboxyl terminus of beta ARK to bind beta gamma subunits, and the ability of synthetic peptides comprised of beta ARK sequences to inhibit beta gamma activation of beta ARK1. We find that the minimal beta gamma binding domain of beta ARK is localized to a 125-amino acid residue stretch, the distal end of which is located 19 residues from the carboxyl terminus. A single 28-mer peptide (Trp643 to Ser670) derived from this sequence effectively inhibited beta gamma activation of beta ARK1, with an IC50 of 76 microM. The identification of this "beta gamma binding domain" on beta ARK and the development of peptide inhibitors provide important tools for the study of G protein-coupled receptor desensitization, as well as for the investigation of beta gamma activation of other G protein-effector systems.
异源三聚体G蛋白的βγ亚基在调节受体刺激的信号转导过程中发挥重要作用。最近在这些作用中发现，它们参与了导致毒蕈碱胆碱能受体（羽贺，K.，和羽贺，T.（1992年）《生物化学杂志》267，2222 - 2227）和β - 肾上腺素能受体（皮彻，J. A.，英格利斯，J.，希金斯，J. B.，阿里扎，J. L.，凯西，P. J.，金，C.，贝诺维奇，J. L.，夸特拉，M. M.，卡隆，M. G.，和莱夫科维茨，R. J.（1992年）《科学》257，1264 - 1267）磷酸化及随后脱敏的信号事件。βγ通过特定的β肾上腺素能受体激酶（βARK） - βγ相互作用，介导β肾上腺素能受体激酶响应受体激活后的膜靶向作用。此过程利用了βγ异戊二烯化γ亚基的膜锚定特性。在本研究中，我们采用了三种不同方法来鉴定βARK羧基末端内与βγ结合从而导致膜易位的区域。我们研究了βγ增强一系列牛βARK1截短突变体酶活性的能力、含有βARK不同长度羧基末端的谷胱甘肽S - 转移酶融合蛋白结合βγ亚基的能力，以及由βARK序列组成的合成肽抑制βγ激活βARK1的能力。我们发现βARK最小的βγ结合结构域定位于一个125个氨基酸残基的片段，其远端距离羧基末端19个残基。从该序列衍生的一个28肽（Trp643至Ser670）有效抑制了βγ对βARK1的激活，IC50为76微摩尔。在βARK上鉴定出这个“βγ结合结构域”以及开发肽抑制剂，为研究G蛋白偶联受体脱敏以及研究其他G蛋白效应系统的βγ激活提供了重要工具。
