The Specific Ligand Screening For M₂-G_i1α Fusion Protein Expressed In Sf9 Cells

PrefaceMuscarinic acetylcholine receptors(MAChRs),is one of the most important receptors and distribute widely in our body.It is included in G protein-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(O₁-O₃).MAChRs are classified to five subtypes according to the typicall structures of i₃ loop which forms anαhelix composed of 157-240 amino acids,the key structure 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(39～46kD),β(36kD)andγ(7～8kD)subunit.αsubunit is the functional subunit,has special amino sequence and structure,varies greatly among each type of G protein.According toαsubunit,G protein can be devided into four types:G_s,G_i,G_q,G_12/13.In the signal transduction mediated by G protein,GPCRs,G protein and effector are basic element,their structure,function and role in sigal transduction are gradually to be discovered.MAChRs is devided into two groups according to their function:M1 group(M₁, M₃,M₅)and M2 group(M₂,M₄).M1 group act on phosphoinosital system to activate phospholipase C(PLC),produce inositol 1,4,5-trisphosphate(IP₃)and 1,2 diacylglycerol(DAG);M2 group inhibit adenylyl cyclase(AC)to reduce cAMP production through coupling to G_i/G_o protein.But there is still doubt when refers to the contrast of detailed coupling mechanism of MAChRs to G_q/11or G_i and G_s.In our study,we recombine the M₂AChR cDNAs and G protein G_ilαcDNAs, express the M₂-G_ilαfusion 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 M₂AChR.Materials and Methods1,MaterialsThe cDNAs of human M₂AChR(PEF-hm2)and bovine G_ilα(pG_α28),baculovirus vector plasmid pBacPAK9 cDNAs,acetylcholine(ACh),atropine,oxotremorine, arecoline,fangchinoline,levitimide and guanosine-5'- O - 3-thiotriphosphate(GTPγS) 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-thiotriphosphate),40PBq/mol) from Amersham pharmacia Co.2,MethodsAmplify the cDNAs of M₂AChRs and G_ilαthrough 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 M₂AChR-G_ilαfusion membrane protein through digestion,homogenizing and centrifuge from cell precipitation.Determine the membrane protein concentration by BCA method,do the [³H]L-quinuclidinyl benzilate(QNB)and[³⁵S]GTPγS binding assay to detect the function of M₂-G_ilαfusion protein and screen the specific ligands for M₂ receptor.ResultsThe cDNAs of M₂AChR and G_ilαis 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 M₂AChR-G_ilαfusion membrane protein,homogenizing and centrifuge from the cell precipitation.The protein concentration is 1.68mg/ml determined by BCA method.The[³H]QNB radioactive ligand binding assay show the fusion protein has M₂ 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 acetylcholine, oxotremorine,arecoline are agonists for M₂ receptor,atropine,fangchinoline, levitimide are antagonists for M₂ 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 M₂AChR-G_ilαfusion protein under the effecting of different ligands,study the molecular interaction and influencing factors in the signal transduction,determine the specific ligands for M₂ receptor.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 G_ilαin the fusion protein and GDP.That is,the lower the affinity is,the lower the capability that GDP dissociate from G_ilαcatalyzed by M₂ receptor-the affinity decreases under the effection of agonists,but increases under that of antagonists.The affinity of GDP is the lowest under the effection of full agonists, lower under that of partial agonist and the strongest under that of antagonists.So we can identify the unknown agonists or antagonists of M₂ receptor by analyzing the affinity of GDP and M₂-G_ilαfusion protein.The successfully expression of M₂-G_ilαfusion protein not only provide an convenient method for studying M₂ receptor and the signal transduction,but also provide path for screening specific ligands for muscarinic recaptor subtype 2,and become a useful system in drug developing and manufacturing of muscarinic acetylcholine receptors.ConclusionThe M₂-G_ilαFusion protein expressed by baculovirus-Sf9 cells expression system have the characterization of binding to ligands and the capability of coupling,the ligands activate the fusion protein through changing the affinity of GDP and G_ilαin the fusion protein.We can screen for the specific agonists and antagonists for muscarinic recaptor.From our study,we conclude that acetylcholine(ACh),oxotremorine and arecoline are agonists for M₂ receptor,atropine,fangchinoline and levitimide are antagonists for M₂ receptor.