Receptor-G^5; fusion proteins as a tool for ligand screening

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• 作者：Guo ; Zheng-Dong ; Suga ; Hinako ; Okamura ; Michiko ; Takeda ; Shigeki ; Haga ; Tatsuya
• 关键词：G protein-coupled receptors ; Muscarinic acetylcholine receptors ; Fusion proteins
• 刊名：Life Sciences
• 出版年：2001
• 出版时间：April 6, 2001
• 年：2001
• 卷：68
• 期：19-20
• 页码：2319-2327
• 全文大小：145 K

文摘

We have prepared fusion proteins of muscarinic M₁- M₅ receptors with ^5; subunits of G proteins G_i1, G_i2, G_s, G₁₁, G₁₆ and chimera of G protein ^5; subunits using the bacurovirus-Sf9 expression system. In fusion proteins such as M₂-G_i1^5; and M₄-G_i1^5;, agonist caused the decrease in the apparent affinity for GDP of these fusion proteins and then the increase in [³⁵S]GTPγS binding in the presence of GDP. Thus we could use the membrane preparation expressing these fusion proteins as a tool to screen agonists and antagonists. On the other hand, the effect of agonists to decrease the apparent affinity for GDP was not clearly observed in fusion proteins of G_q/G₁₁-coupled receptors such as M₁-G₁₁^5;, M₃-G₁₁^5;, and M₅-G₁₁^5;. The effect of agonists could be observed for fusion proteins with G₁₆^5; of muscarinic M₁, M₂ and adrenergic β2 receptors, but the extent of the effect was much less than that for fusion proteins with G_i1^5; of G_i/G_o-coupled receptors. Fusion proteins of M₁ receptors with G_i1^5; or chimera of G₁₆^5; and G_i2^5; were also not effective in detecting the action of agonists.