| ObjectiveMuscarinic acetylcholine receptors ( mAChRs ) is a member of G-protein-coupled receptors (GPCRs) superfamily, which have characteristic molecular structure and signal transduction method. Activation of the GPCRs by extracellular ligands induces the exchange of GDP with GTP on G-protein, leading to the dissociation of Ga-GDPBy complex into Ga-GTP and GBy which activates effectors respectively. The transmembrane signal transduction system contained mAChR , G-protein and effector as well: m,/m3/m5 sub types couple to Gqa/G0 a, activating downstream effector-PLC, while m2/m4 subtypes couple to Gia, inhibiting the effector-AC. At present the GPCR-G protein fusion protein used for GPCR molecular analysis has three main advantages: (1) the defined 1:1 stoichiometry of the signaling partners; (2) the close physical proximity of the signaling partners; and (3) the tight tethering of Gato the membrane. We constructed m4AchR and Gilafusion protein- m4AchR-Gila and analyzed its ligand - binding functional characters and some factors impaction on m4AchR and G-protein coupling.Materials and MethodsMaterials - The cDNAs of human m4AchR and bovine Gila and baculovir-us (pBacPAK9) ; Sf9 insect cells; IPL-41 insect culture media; Fetal bovine serum; Tryptose phosphate broth pluronic F-68 solution; [3H]QNB and [35S] GTPrS.Methods-for generation of m4 AchR-Gilofused cDNAs: in two PCR reactions, the DNA sequence of the C-terminus of the m4AchR was linked with the N-terminus of Gila. Using baculovirus-insect-cell ( Sr9 cells) system expressed m4AchR protein and m4AchR-Gila fusion protein. [ 3H] QNB saturation binding in Sf9 membrane was performed to determine the expression level of m4AchR-Gilaand m4AchR. High affinity [ 35S] GTP-yS binding was produced to detect the effect of various m4AchR ligands or different concentration of Na + and K+ on [35 S]GTPrS binding in the presence of GDP, GTP and ATP.Results1. The construction of pBacPAK9-m4AChR-GilacDNAs expression vectorsWe have xonstructed pBacPAK9-m4 AChR-GilacDNAs expressing vectors by two PCR, and the results of DNA sequence analysis showed that target DNAs in Baculovirus vector plasmid ( pBacPAK9) were m4AChR-Gilafused cDNAs.2. The expression of m4AChR-Gilafusion proteinWe have expressed the m4AChR-Gila fusion protein successfully in Sf9-in-sect cells.3. Determination of m4AChR protein and m4 AChR-Gila fusion protein expression levelThe expression level of isolated m4AChR protein and m4AChR-Gila fusion protein expressed in Sf9 cells membrane were 41.91 3.06 pmol . mg-1 protein and 47. 04 1. 58 pmol. mg-1 protein based on the [ 3H] QNB bound (table 1).4. GTP-/S reducing [3H]QNB binding on m4AChR-Gila fusion protein Ligand-directed GTP-yS binding to G-protein is a sensitive method for research on GPCR-G-protein coupling. Fig. 1A and B show that the increasing of [3H]QNB concentration the quantity of bound [3H]QNB on m4AChR and m4 AChR-Gila were both climbing to the highest level at about 10nmmol . L-1[3H] QNB existing, which indicated that both the isolated m4AChR and m4 AChR-Gila fusion protein had the pharmacological character of muscarinic receptor. With 10uM GTPrS, the [3H]QNB binding was decreased on m4AChR-Gila fu-sion protein while not being influenced on m4AChR.5. [35 S ] GTP-yS binding studiesFig. 2 and Fig. 3 summarized the [35S]GTP-yS binding on m4AChR and m4AChR-Gila fusion protein in the presence of full agonist acetylcho-line, carbamylcholine, antagonist prifinium and atropine . On m4 AChR-Gila fusion protein, acetylcholine and carbamylcholine induced the largest bound [35S] GTP-yS amount among these four ligands, carbamylcholine took the second place, and prifinium and atropine are the last. But the were all higher than the binding of [35S]GTP-yS on m4AChR, moreover, no significant difference could be detected among the same four ligands.6. Effects of full agonist, partial agonist or antagonist on displacement curves by GDP, GTP or ATP of [35S]GTPyS bindingFig. 4 shows the displacement...
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