| PrefaceMuscarinic acetylcholine receptors(MAChRs)is one of the most important receptors and distribute widely in our body.It is included in G prtein-coupled receptors (GPCRs)family,a glucoprotein with molecular weight of 51-66Kd,composed of 460-590 aminoacids,sharing the common structure with other GPCRs—a seven transmembrane from the extracellular N terminal to the intracellular C terminal, forming 3 intracellular loop(i1-i3)and 3 extracellular loop(Oi-O3).MAChRs are classified to five subtypes according to the typicall structures of i3 loop which forms an ahelix composed of 157-240 amino acids,the key structures for receptor action. MAChRs distribute widely in central and peripheral nervous system,heart,smooth muscle,glands,trigger endocellular signal transduction and biochemistry reaction through binding to exogenous or endogenous ligands as a transmembrane protein,and take part in the process of disease.G protein-Guanine nucleotide-binding protein,which possess GTPase activity,can hydrolyse GTP to GDP and switch on and off according to the binding of GTP or GDP. G protein is a very important signal molecule with the molecular weight of about 100KD,composed ofα(3946KD),β(36KD),γ(78KD)subunit.asubunit is the fuctional subunit,has special amino sequence and structure,varies greatly among each type of G protein.According toasubunit,G protein can be devided into four types:Gs,Gl,Gq,G12/13.In the signal transduction mediated by G protein,GPCRs,G protein and effector are basic element,their structure,function and role in signal transduction are gradually to be discovered.MAChRs is divided into two groups according to their funtion:M1 group(composed M1,M3,M5)and M2 group(composed M2,M4).Ml group act on phosphoinosital system to activate phospholipase C(PLC),produce inositol l,4,5,-trisphosphate(IP3)and 1,2diacylglycerol(DAG);M2 group inhibit adenylyl cyclase(AC)to reduce cAMP production through coupling to Gi/Go protein.But there is still doubt when refers to the contrast of detailed coupling mechanism of MAChRs to Gq/11or Gi and Gs.In our study,we recombing the M3AChR cDNAs and G protein G11αcDNAs, express the M3-G11αfusion protein in Sf9 insect cells by baculovirus expression system, then detect the function of the fusion protein to make clear the interaction mechanism and affective factors using radioactive ligand binding experiment,screening for the effective drugs of M3AChR.Materials and Methods一,MaterialsThe cDNAs of human M3AChR and bovine G11α,baculovirus vector plasmid pBacPAK9 cDNAs was generously provided by prof.T.Haga,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;TNM-FH insect cell culture media was bought from sigma company;fetal bovine serum was bought from TBD company; [3H]QNB(L-quinuclidinyl benzilate,1.8pBq/mol)and[35S]GTPγs(guanosine-5'-O-(3-thiotriposphate), 40pBq/mol)from Amersham pharmacia Co.acetycholine(ACH), atropine(Atro),pilocarpin(Pilo),Carbacholine,4-damp,MCN-A-343,Dafinaxin(Dafi) and guanosine-5'-O-3-thiotriphosphate(GTPγS)were bought from sigma company.二,MethodsAmplify the cDNAs of M3AChR and G11αthrough PCR reaction,cut down the right band on electrophoresis gel and purify it for the linkage by the second step PCR. Cut down the connected band,purify again.Digest the cDNAs and insert into pBacPAK9 vector,transfect the Sf9 cells,collect the cells on the second day after transfection,extract the virus and harvest the cells.Obtain the M3-G11αfusion membrane protein through digestion,homogenizing and centrifuge from cell precipitation.Determine the membrane protein concentration by BCA method,do the [3H]QNB(L-quinuclidinylbenzilate),[35S]GTPγS Guanosine5'O-(γ-Thio)Triphosphate binding assay to detect the function of M3-G11αfusion protein and screen the specific ligands for M3 receptor.ResultsThe cDNAs of M3AChR and G11αis amplified respectively then insert into baculovirus vector and expressed successfully in Sf9 insect cells.The Sf9 cells become larger,uneven and corpuscular after transfection.Collect the cells the second day after transfection,extract the virus and harvest the cells.Obtain the M3-G11αfusion membrane protein,homogenizing and centrifuge from the cell precipitation.The protein concentration is 2.86mg/ml determined by BCA method.The[3H]QNB radioactive ligand binding assay show the fusion protein has M3 receptor character that the high specific,high level binding site,and the binding level rise as the increase of the concentration of[3H]QNB.[35S]GTPγS binding assay prove that Ach,Pilo are completely agonists for M3 receptor;Cch,MCN-A-343 are partial agonists for M3 receptor;Atro,4-Damp,Dafi are antagonists for M3 receptor.DiscussionThe rapidly developed biomolebular technique provided new methods in the research of muscarinic receptor and signal transduction.In our research,we observe the changes of expression and function of high efficient M3-G11αfusion protein under the effecting of different ligands,study the molecular interaction and influencing factors in the signal transduction,determine the specific ligands for M3 receptor.[35S]GTPγS binding to G protein regulated by ligands is a sensitive method for studying coupling of muscarinic acetylcholine receptor and G protein.In the binding experiment substitute GDP for[35S]GTPγS,the changes of[35S]GTPγS binding effection demonstrate that the direction and degrees of the curves varies under different effections of various ligands and different concentrations of GDP.These differences stand for the affinity of G11αin the fusion protein and GDP.That is,the lower the affinity is,the lower the capability that GDP dissociate from G11αcatalyzed by M3 receptor-the affinity decreases under the effection of agonists,but increases under that of antagonists.The affinity of GDP is the strongest under that of reverse agonists.So we can identify the unknown agonists or antagonists of M3 receptor by analyzing the affinity of GDP and M3-G11αfusion protein.The successfully expression of M3-G11αfusion protein not only provide an convenient method for studying M3 receptor and the signal transduction,but also provide path for screening specific ligands for muscarinic receptor subtype 3,and become a useful system in drug developing and manufacturing of muscarinic acetylcholine receptors.ConclusionThe M3-G11αfusion protein expressed by baculovirus-Sf9 cells expression system have the characterization of binding to ligands and the capability of coupling,the liangds activate the fusion protein through changing the affinity of GDP and G11αthe fusion protein.We can screen for the specific agonists and antagonists for muscarinic receptor.From our study,we conclude that Ach,Pilo are completely agonists for M3 receptor;Cch,MCN-A-343 are partial agonists for M3 receptor;Atro,4-Damp,Dafi are antagonists for M3 receptor.
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