| As one of the superfamily of G-protein coupled receptors ( GPCRs) , musca-rinic acetylcholine receptor (mAchR) shows some typical characteristics of the molecular structure and signal transduction in this family. That extracellular ligands activate GPCRs may induce the exchange between GDP and GTP on G-protein, leading to the dissociation of Ga-GDP complex into Ga-GTP and G that will activate concequently their effectors. The transmembrane's signal transduction system consist of mAchR, G-protein and effectors. The subtypes of m1 m3, m5 can couple with Gqa/Goa, activating downstream effector-PLC, while the other subtypes of m2, m4 connect with Gia, inhibiting adenylate cyclase (AC). Up to now, the GPCR-G fusion protein used for molecular analysis of GPCR has embodied three main strengths: (1) the exact 1: 1 stoichiometry of the signaling partners; (2) the close physical proximity of the signaling partners ; and (3 ) the tight tethering of Got to the membrane. We constructed m2AchR and Gila fusion protein (m2AchR-Gilci) and analyzed its ligand-binding characteristics and some factors impacting on m2AchR and G-protein coupling.Materials and MethodsMaterialsThe cDNAs of human m2AchR,bovine Gila and baculovirus (pBacPAK9) is generously provided by Prof. Haga. Baculovirus-insect-cells ( Sr9) is a beneficence kindly donated by Prof. Chen Jianguo. The insect culture media of IPL-41 is made in JRH Biosciences. Fetal bovine serum, tryptose phosphatebroth,pluronic F-68 solution are all made in Sigma. [3H] QNB (48. 0 Ci/ mmol) , [35S]GTPS (1084 Ci/mmol) are from Amersham Pharmacia Biotech.MethodsFor generation of m2AchR-Gila fusion protein,the DNA sequence of the C terminus of the m2AchR was linked with the N terminus of Gila in two steps of PCR reactions. The isolated m2AchR protein or m2AchR-Gila fusion protein can be expressed under the surpport of Sf9 expression system. [3H] QNB saturation binding in Sf9 membranes is performed to determine the expression level of m2AchR protein or m2AchR-Gilct fusion protein. High affinity [35 S ] GTPS binding detects the effect of various m2AchR ligands on [35S] GTPS binding in the coexistence of GDP, GTP or ATP.Result1. Examination of the expression level of m2AchR protein and m2AchR-Gila fusion proteinBased on the [3 H ] QNB binding experiments ( table 1) , the expression levels of isolated m2AchR protein and m2AchR-Gila fusion protein in Sf9 cell membranes are 13.45 0.25 pmol/mg protein and 18.14 0. 17 pmol/mg protein.2. GTPS reducing [3 H]QNB binding to m2AchR-Gila. fusion protein Ligand-regulated GTPS binding to G proteins is a sensitive method forstudying GPCR-G protein coupling. Fig. 3 shows that, with the increase of [3H] QNB concentration, the quantity of bound [3 H ] QNB on m2 AchR protein and m2AchR-Gila fusion protein are both climbing gradually to reach the top on the condition of 10nM [3H] QNB , which indicates that both of the isolated m2AchR protein and m2AchR-Gila fusion protein have pharmacological characteristics of muscarinic receptors. In the situation of 10mM GTPS, [3H]QNB binding can decrease on m2 AchR-Gila. fusion protein while not being influenced on m2AchRprotein.3. Exmination for [35S]GTPS bindingTable. 2 summarizes the [ 5 S ] GTPS binding on m2AchR protein and m2AchR-Gila fusion protein in the existence of agonist Ach and Cch,or antagonist Atropine and AF-DX116 without GDP. On m2AchR-Gila fusion protein, Ach and Cch induce a 1 arge amount of bound [35 S ] GTPS whereas Atropine and AF-116 do a bit. But they are all higher than [35S] GTPS binding on m2AchR protein does,in which the significances cannot be detected among the same four ligands. Fig. 4 shows the similar trend of [35S]GTPS binding.4. Effects of agonists or antagonists on displacement curves from GDP, GTP or ATP of [35S] GTPS bindingFig. 4 shows the displacement curves from GDP of [35S]GTPS binding on the condition of diversities of muscarinic ligands of m2 AchR-Gila fusion protein. The displacement curves move toward the right on the...
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