Tsu R C, Ho M K, Yung L Y, Joshi S, Wong Y H
Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon.
Mol Pharmacol. 1997 Jul;52(1):38-45. doi: 10.1124/mol.52.1.38.
Many Gi-coupled receptors are known to interact with the pertussis toxin (PTX)-insensitive Gz protein. Given that the alpha subunits of Gi and Gz share only 60% identity in their amino acid sequences, their receptor-interacting domains must be highly similar. By swapping the carboxyl termini of alpha i2 and alpha z with each other or with those of alpha t, alpha12, and alpha13, we examined the relative contributions of the carboxyl-end 36 amino acids of the alpha chains toward receptor recognition. Chimeric alpha chains lacking the site for PTX-catalyzed ADP-ribosylation were coexpressed with the type II adenylyl cyclase (AC II) and one of several Gi-coupled receptors (formyl peptide, dopamine D2, and delta-opioid receptors) in human embryonic kidney 293 cells. The alpha i2/alpha z chimera was able to interact with both aminergic and peptidergic receptors, resulting in betagamma-mediated stimulation of AC II in the presence of agonists and PTX. Functional and mutational analyses of alpha i2/alpha z revealed that this chimera can inhibit the endogenous ACs of 293 cells. Similarly, the alpha z/alpha i2 chimera seemed to retain the abilities to interact with receptors and inhibit cAMP accumulation. Fusion of the carboxyl-terminal 36 amino acids of alpha z to a backbone of alpha t1 produced a chimera, alpha t1/alpha z, that did not couple to any of the Gi-coupled receptors tested. Interestingly, an alpha13/alpha z chimera (with only the last five amino acids switched) displayed differential abilities to interact with receptors. Signals from aminergic, but not peptidergic, receptors were transduced by alpha13/alpha z. A similar construct, alpha12/alpha z, behaved just like alpha13/alpha z. These results indicated that "alpha i-like" or "alpha z-like" sequences at the carboxyl termini of alpha subunits are not always necessary or sufficient for specifying interaction with Gi-coupled receptors.
已知许多与Gi偶联的受体可与百日咳毒素（PTX）不敏感的Gz蛋白相互作用。鉴于Gi和Gz的α亚基在氨基酸序列上仅有60%的同源性，它们与受体相互作用的结构域必定高度相似。通过将αi2和αz的羧基末端相互交换，或与αt、α12和α13的羧基末端交换，我们研究了α链羧基末端36个氨基酸对受体识别的相对贡献。缺乏PTX催化的ADP核糖基化位点的嵌合α链与人胚肾293细胞中的II型腺苷酸环化酶（AC II）以及几种与Gi偶联的受体（甲酰肽、多巴胺D2和δ阿片受体）之一共表达。αi2/αz嵌合体能够与胺能和肽能受体相互作用，在激动剂和PTX存在的情况下，导致βγ介导的AC II激活。对αi2/αz的功能和突变分析表明，该嵌合体能够抑制293细胞的内源性AC。同样，αz/αi2嵌合体似乎保留了与受体相互作用和抑制cAMP积累的能力。将αz羧基末端的36个氨基酸融合到αt1的主干上产生了一种嵌合体αt1/αz，它不与任何测试的与Gi偶联的受体偶联。有趣的是，一种α13/αz嵌合体（仅最后五个氨基酸交换）表现出与受体相互作用的不同能力。胺能受体而非肽能受体的信号由α13/αz转导。一种类似的构建体α12/αz的行为与α13/αz相同。这些结果表明，α亚基羧基末端的“αi样”或“αz样”序列对于指定与Gi偶联受体的相互作用并非总是必要或充分的。
