Kostenis E, Conklin B R, Wess J
Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA.
Biochemistry. 1997 Feb 11;36(6):1487-95. doi: 10.1021/bi962554d.
The molecular basis of receptor/G protein coupling selectivity was studied by using the m2 muscarinic receptor, a prototypical G(i/o)-coupled receptor as a model system. We could recently show that the m2 receptor can efficiently interact with mutant G protein alpha(q) subunits in which the last five amino acids were replaced with alpha(i2) or alpha(o) sequence [Liu, J., Conklin, B. R., Blin, N., Yun, J., & Wess, J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11642-11646]. Additional mutagenesis studies led to the identification of a four-amino-acid motif on the m2 receptor (Val385, Thr386, Ile389, and Leu390) that is predicted to functionally interact with the C-terminal portion of alpha(i/o) subunits. To further investigate the structural requirements for this interaction to occur, these four m2 receptor residues were replaced, either individually or in combination, with the corresponding residues present in the G(q/11)-coupled muscarinic receptors (m1, m3, and m5). The ability of the resulting mutant m2 receptors to interact with a mutant alpha(q) subunit (qo5) in which the last five amino acids were replaced with alpha(o) sequence was investigated in co-transfected COS-7 cells [studied biochemical response: stimulation of phosphatidyl inositol (PI) hydrolysis]. Our data suggest that the presence of three of the four targeted m2 receptor residues (Val385, Thr386, and Ile389) is essential for efficient recognition of C-terminal alpha(i/o) sequences. To study which specific amino acids within the C-terminal segment of alpha(i/o) subunits are critical for this interaction to occur, the wild type m2 receptor was co-expressed with a series of mutant alpha(q) subunits containing single or multiple alpha(q) --> alpha(i1,2) point mutations at their C-terminus. Remarkably, the wild type m2 receptor, while unable to efficiently stimulate wild type alpha(q), gained the ability to productively interact with three alpha(q) single-point mutants, providing the first example that the receptor coupling selectivity of G protein alpha subunits can be switched by single amino acid substitutions. Given the high degree of structural homology among different G protein-coupled receptors and among different classes of G protein alpha subunits, our results should be of broad general relevance.
通过使用M2毒蕈碱受体（一种典型的G(i/o)偶联受体）作为模型系统，研究了受体/G蛋白偶联选择性的分子基础。我们最近发现，M2受体可以与突变型G蛋白α(q)亚基有效相互作用，其中最后五个氨基酸被α(i2)或α(o)序列取代[刘，J.，康克林，B.R.，布林，N.，云，J.，&韦斯，J.（1995年）美国国家科学院院刊92，11642 - 11646]。进一步的诱变研究导致在M2受体上鉴定出一个四氨基酸基序（Val385、Thr386、Ile389和Leu390），预计该基序与α(i/o)亚基的C末端部分发生功能相互作用。为了进一步研究这种相互作用发生的结构要求，将这四个M2受体残基单独或组合地替换为G(q/11)偶联的毒蕈碱受体（M1、M3和M5）中存在的相应残基。在共转染的COS - 7细胞中研究了所得突变型M2受体与突变型α(q)亚基（qo5）相互作用的能力，其中最后五个氨基酸被α(o)序列取代[研究的生化反应：磷脂酰肌醇（PI）水解的刺激]。我们的数据表明，四个靶向的M2受体残基中的三个（Val385、Thr386和Ile389）的存在对于有效识别C末端α(i/o)序列至关重要。为了研究α(i/o)亚基C末端片段中的哪些特定氨基酸对于这种相互作用的发生至关重要，将野生型M2受体与一系列在其C末端含有单个或多个α(q)→α(i1,2)点突变的突变型α(q)亚基共表达。值得注意的是，野生型M2受体虽然不能有效刺激野生型α(q)，但获得了与三个α(q)单点突变体有效相互作用的能力，这提供了第一个例子，即G蛋白α亚基的受体偶联选择性可以通过单个氨基酸取代来切换。鉴于不同G蛋白偶联受体之间以及不同类别的G蛋白α亚基之间高度的结构同源性，我们的结果应该具有广泛的普遍相关性。
