Identification of specific drugs for M_5 using m_5AChR-G_ (11?) fusion protein
- VernacularTitle:应用m_5AChR-G_(11?)融合蛋白鉴别M_5受体亚型的特异性药物
- Author:
Hongrui LIU
;
Zhengdong GUO
;
Xuesong HAN
;
Haibo WANG
;
Zhi LI
- Publication Type:Journal Article
- Keywords:
muscarinic acetylcholine receptor;
G protein;
fusion protein;
agonist;
antagonist
- From:
Chinese Pharmacological Bulletin
2003;0(08):-
- CountryChina
- Language:Chinese
-
Abstract:
Aim The m_5AChR-G_ 11? fusion protein was expressed by baculovirus-Sf9 cells system, then using it to identify the specific agonists and antagonists for m_5AChR via detecting the affinity of GDP and m_5AChR-G_ 11?. Methods The m_5AChR-G_ 11? fused cDNAs were generated via a two-step PCR protocol and inserted into pBacPAK9 virus vector. We expressed m_5AChR-G_ 11? fusion protein and m_5AChR protein using baculovirus-Sf9 cell system. [ 3H]QNB and [ 35S]GTP?S binding tests were performed to detect the expressional level of receptor proteins and determine the affinity of GDP and m_5AChR-G_ 11? fusion protein. Results The expression level of m_5AChR-G_ 11? was (47.6?3.2) nmol?g -1 protein. The affinity of GDP to G_ 11? partner changed in the presence of different muscarinic ligands. IC_ 50 values of GDP in the presence of ACh, YM796, Oxotremorine, Methixene, Dextimide and atropine were 128.0, 72.1, 68.5, 16.2, 14.9 and 9.7 ?mol?L -1 respectively, and that in the absence of muscarinic ligand was 20.8 ?mol?L -1. Conclusion The m_5AChR-G_ 11? fusion protein has the pharmacological specificity of M_5 receptor and the efficient coupling interaction of the two partner. Affinity of GDP to ligand bound m_5AChR-G_ 11? fusion protein represents the species of muscarinic ligands. ACh is a full agonist for m_5AChR-G_ 11? fusion protein, YM796 and oxotremorine are partial agonists, while methixene, dextimide and atropine are antagonists.
