Effects Of Various Muscarinic Ligands On M₁AChR-G₁₁α Fusion Protein Expressed In Bac-to-Bac Baculovirus Expression System

prefacemAChRs are members of a superfamily of GPCR.Five subtypes of mAChR (m₁-m₅)have been identified by cloning of cDNA or genes and by their expression in cultured cells.The structure of mAChR has the characteristic of GPCR,which are composed of seven transmembrane regions(TM_1-7),three extracellular loops(O_1-3), three intracellular loops(i_1-3),N-terminal outside the cells,and C-terminal inside.The "odd-numbered" muscarinic receptors(m1,m3,and m5)typically couple via the subunits of the G_q/11family,where the "even-numbered" members(m₂,m₄)couple via G_i and G_o subunits.Fusion protein between GPCR and G proteins is a novel means to examine the functional interactions of a GPCR with a single G protein.The most salient properties of GPCR-Gαfusion proteins are:the defined 1:1 stoichiometry of the signaling partners;the close physical proximity of the signaling partners;and the tight tethering of Gαto the membrane.The aim of our study was to generate a fusion protein of m₁AChR and G₁₁αin Bac-to-Bac baculovirus expression system and analyze the effects of various muscarinic ligands on the interaction of fused m₁AChR and G₁₁α. Materiales and Methods1.MaterialsThe cDNAs of human m₁ subtype(PEF-hm₁),bovine G₁₁α(G12α),pFastBac1, E.coli DH5α,E.coli DH10Bac(contain Bacmid and Helper plasmids)generously provided by Prof.HAGA Tatsuya(Institute of Biomolecular Science,Gakushuin University,Tokyo,Japan);Sf9 insect cells were kind gifts from Prof.CHEN Jian-Guo (Department of Cellular Biology,Institute of Biosciences,Beijing University,Beijing, China);Axygen gel extract kit,fetal bovine serum,and tryptose phosphate broth were purchased from Sigma Chemical Co;[³H]QNB,and[³⁵S]GTP_γS were from Amersham Pharmacia Company;all other reagents were of the highest purity and available from standard suppliers.2.MethodsThe amplification of m₁AChR and m₁AChR-G₁₁αDNA:m₁AChR-G₁₁αDNA was generated by a two-step PCR protocol using Taq polymerase.In PCR 1A,the DNA sequence of m₁AChR was amplified with PEFhm₁ as template using a sense primer (m₁-BamHâ… )and an antisense primer(m₁-C end).In PCR 1B,the DNA sequence of G₁₁αwas amplified with G12αas template using a sense primer(m₁AChR-G₁₁α)and an antisense primer(G₁₁α-C end).In PCR 2,the DNA fragments from PCR 1A and PCR 1B were anneled and amplified using the sense primer of PCR 1A and the antisense primer of PCR 1B.The fragments from PCR 2 were digested with BamHâ… and Salâ… and cloned into pFastbacl digested with BamHâ… and Salâ… .PCR generated DNA sequences were confirmed by restriction enzyme analysis and enzymatic sequencing.Generation of recombinant baculovirus,cell culture,and membrane preparation: Recombinant baculovirus was generated in Bac-to-Bac baculovirus expression system. Sf9 cells were homogenized in Dounce and centrifuged.Pellets were resuspended in 20mmol·L^-1Hepes-KOH(pH=8.0),containing 1mmol·L^-1EDTA,2mmol·L^-1MgCl₂, 0.5mmol·L^-1PMSF,5μg·ml^-1leupeptide,5μg·ml^-1pepstain,5mmol·L^-1benzamidine, and stocked in aliquots at -80℃until use.[³H]QNB and[³⁵S]GTP_γS binding assays:Before experiments,membranes were resuspended in HEN buffer.Sf9 membranes expressed m₁AChR and m₁AChR-G₁₁αfusion proteins were suspended in 100μl HEN buffer supplemented with[³H]QNB.In the standard assay for[³⁵S]GTP_γS binding,the membrane preparations were incubated with[³⁵S]GTP_γS 0.3 nmol·L^-1and different concentrations of GDP in the presence of various muscafinic ligands.Incubations were performed for 60 min at 30℃.The reactions were terminated with 2.5 ml cold Na⁺-K⁺ buffer.Bound[³H]QNB or [³⁵S]GTP_γS was trapped on glass fiber filter paper,washed three times with 2.5 ml cold Na⁺-K⁺ buffer,and then counted with Beckman liquid scintillation counter.ResultsBoth m₁AChR and m₁AChR-G₁₁αfusion protein were expressed in Sf9 cells.And the expression level was 24.03±2.13 and 45.39±2.16 nmol/g protein respectively, assessed by saturation analysis of specific binding of the muscarinic receptor antagonist [³H]QNB.Ligand-regulated GTP_γS binding to G proteins is a sensitie method for studying GPCR-G protein in coupling.Addition of the muscarinic receptor agonist ACh to membranes expressed m₁AChR did not result in activation of endogenous G₁₁αproteins as measured by the extent of[³⁵S]GTP_γS binding,and no significant difference was detected among ACh and atropine,howere,ACh activated m₁AChR-G₁₁αfusion protein which induced significant elevation of bound[³⁵S]GTP_γS.The affinity of GDP to G₁₁αprotein partner changed in the presence of different muscarinic ligands,IC₅₀value of GDP in the presence of ACh,CCh,AF-267B, tiotropium bromide,telenzepine and atropine were 131.82,61.66,28.18,3.24,6.02 and 5.25μmol·L^-1,respetively,and that in the absence of muscarinic ligand was 16.60μmol·L^-1.Conclusions1.We can successfully express m₁AChR and m₁ChR-G₁₁αfusion protein in Bac-to-Bac baculovirous expression systerm.2.m₁AChR-G₁₁αfusion protein is useful to study the signal transduction of m₁AChR.