Preparation of m_1AChR-G_(11)fusion protein and m_4AChR-G_(16)fusion protein and effects of various ligands on them
- VernacularTitle:m_1AChR-G_(11)和m_4AChR-G_(16)融合蛋白的建立、表达及几种配体的作用
- Author:
Haibo WANG
;
Zhengdong GUO
;
Haipeng ZHANG
;
Juan WANG
;
Hua WEN
;
Xinmin LI
- Publication Type:Journal Article
- Keywords:
Receptors, muscarinic;
Signal transduction;
Ligand;
G-protein
- From:
Chinese Journal of Pathophysiology
2000;0(12):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To prepare m 1AChR-G 11 and m 4AChR-G 16 fusion protein in Baculovirus-Sf9 cell system and detect the effects of various muscarinic ligands on the interaction between m 1AChR and G 11 and m 4AChR and G 16 ,and screen different kinds of ligands specific for m 1 and m 4. METHODS: To prepare fused DNA of m 1AChR-G 11 and m 4AChR-G 16 in two PCR, then expressed in Sf9 cells and detect the pharmacological function of m 1AChR-G 11 fusion protein and m 4AChR-G 16 fusion protein by [ 3H]QNB and [ 35 S]GTP?S binding experiments; To expore the way of the activation of m 1AChR-G 11 and m 4AChR-G 16 fusion protein by various ligands includingcetylcholine (ACh), Pilocarpine (Pilo), 4-hydroxy-2-butynyl-1-trimethylammonium-m-chloro-carbanilatechloride (McN-A-343),tetrandrine, pirenzepine (PZ), alcuronium, atropine,R-(+)-hyoscyamine and gallamine by displacement by GDP on [ 35 S]GTP?S binding experiments. RESULTS: The expression levels of m 1AChR-G 11 and m 4AChR-G 16 fusion protein were (45 39?2 62) nmol?g -1 protein, (47 04?1 58) nmol?g -1 protein. The affinity of GDP to G 11 and G 16 partner changed in the presence of different muscarinic ligands. CONCLUSION: The m 1AChR-G 11 and m 4AChR-G 16 showed the pharmacological specificity to m 1 and m 4 receptor and the efficient signaling of the two partners. Ligands of m 1AChR and m 4AchR mediated different signal transduction by changing the affinity of G 11 /G 16 and GDP. So m1AChR-G 11 fusion protein and m 4AChR-G 16 fusion protein can be taken as a tool to screen ligands specific for m 1AChR and m 4AChR.
