Ktn Ecr Pcrgamon Frcnilm +aa Frinkdin Glsal 0006.2952(94100294-0 EXPRESSION OF A CANNABINOID RECEPTOR IN BACULOVIRUS-INFECTED INSECT CELLS DEYISE DovE PETTIT , VINcFXT M. SHOwALTER MARY ABOOD Gur CAHRAL+ Microbiolocx(Immunalaz  and *Pharmacology Toxicologr , Depattncnts Commenucalh Univeesity, Medicul Collcgc Mineinia  Vrelu_A Richmond, VA 23298-0678. USA Februars 1924; ecceptd Mey 1594) [Reciued Abslract cannabinoid [eceplor recumbinani buculovins (AcNPV-THCR) has bcen conslrucied JIUJ Norzhern analysis ol [Olel RNAIrom Spodo Iera emploted eiprceS0t ncun)l cnnn-cinoid Tccsciors hyPct-Pro Juctior of j)3kb Inxcl cclls inlecled with AcNPV THICR revcakd notr] Acufiverud  Si cannabin vid [Cccpor cDNA Optimal viral uranschci Dinca probc [ mIcE-amslaled Mc niorcin ~ntctabolically Iabeled AcVPV THCR-infecied St9 cclka cxaression "s cheetdd multplacit" 2,5 , Tvensrrissior THCR-infecicd Si9 ccl> showed cNFV amaccnn n cleclrun Mictucunt cxicosl jucinGTMC per Lrbilion and ele ztron densi cMonlasmic Drtinucic? accumnulativn Indicatine cCcnio Zircontoicin cxniceion inmnunonuonescence suiminz using wnllctum prouuced (usicn Inc cannabinoid ctolcir conskstinz cricinai damain fcccpLor Jnd ncpzllis Anecm ~THCR-infcctcd Sf? cclls cannabinoid AcNPy Ecueaucd Icccplor #IpTeS1CT Scaichanur Rurnthil ['HJCPSS , birding indizaicd Kol JnMand j emol/mg anihys JJIU ICccptor E4u11 immunoblotting AcNPV-THCR-inlected Stv ccll Iyeatcs rcvcaki Wuestern nqulcin Deficlmcd molcculat #cights anging Iron Immunorc12042 band; Win rclativc ZokDa neeuonimam{ (55 KDa) exhibited 2 rclativc molccul "ecics #cial Consisle IL Kh thal predicted Jor th? Iranslational Icomi thc cannabinoid cDNA coding nroduzh obtaincd Tleenar Eenucncg Tanealun UWnE Tnte AcNPVTHCR MRNA also S5 kDa immunorcictive snecies; hickcd dala Indicile Ihal tkc quantitics lotgs birulotins cxpfesslon Ssicmi Is 4 Yoblc mcons cxdicssina aclalvcl cannabincid Icomhinan Feccniar prolci bindins; inoid feceptor: Wads Tadeulinand cannal nfalcin-couplco (cccpOr , Duculovm? crficssion ssicm; Celta-9-Ictrahydrocannabinol: amti canaabinuid receptor anliserum THC:, major psychoactive signal transduction pathways. stimulate inhibit comconcn miarijuana multiplicity enzymes, andalter Tcmbranc can produce eflects In mcmoranc-associaico perccption . including altcrations chanucls . Howevct, numans niood A 4ciaico stcrco psychomotor ac;ivity, cognition, specilicity and memon Waa5 or physio - KatCm Icqwtenienis amliemesis,andimmiunosuppression [1 . 2]. thal gesia logical activity suezest the cannabinoids also mcchanism responsible unlikely that onc specilic receplor [6-10] cal a01 Cannabinoid inducedinhibition ofcAMI' accumu- the   multiplicity THC ccc onserieo uith exposure Indeed, substanlial evidence exis'$ for two lation neuroblastoma ccll Jinc , NISTG2 , mouSC possible mcchanisms THC induced cellular the presence Lelc"  lirs[delinilive evidence effects. The highly lipophilic   nalure of THC has [eceplots [": cunn pinoio neunal esua suegested Radioligand binding studies that utilized ol  thc cannabinold-induced cmt potent perturbation [3-5] aniloz utthz ellects tritiarcd THC CFS5,910. meMbrane Such  disruption of cellular membranes may alter of cannabinoid rcccplors in canbd Rzjsence tissuC Antomical distnbution or the Ticun7 cannabinoid Icceptor in rat brain #1$ characierized Corresponding Dc; Guy Cacnal Mddicl aunor using |HJCPSS,9410, Colleesints autoradiography [ccentor Wiieinij Aezini Commonteill Univeri. Box MCV Station VA 23298-0678, binding observed in cerebellum Richmond cicnsc 825-2306: FAX (5u4) 823-99+6. hppocampus basal ganglia [13,14 The TKC Atorcsiations: dalte-O-tetrehudrccannnbol: screndipitous discovery isolated ckinc cDNA AcNPV calfforaica Amlofrplia mullpi cnvclopcd troin cDNA library , shown ral cetehral corer polyhedrosis Gol nucet ACNPV We[oSals bind and  (unclionally Inicracr Cannalinoids blocking THCR, recombininl bacuiovinus tulict fur the  miolecular ahceseo chaclenizillion cannah naid feceptor; AcNPV-THCR naouolu (cccdl this receptor [s, 1S]. Sequencc analysis of this clone GPCR Iecombinant Oaculovirus Dfolci-Coup_cd high Jevel homolozy with GPCR Iercued HB-Ag.hcpatilis B core anligen: HBcAg THCR, Icceplor; tnar thal mesesses contaimg erten? dnmain hepattis 8 cofc onmeencannioinoidicccpronisonprolcim: MOI , multiplicilg olinlecticn;PMSF . phenylm:tkylsulfoayl Icos aliun slcs even consen Lranememntane and an unletnal or cyloplasmic domain thal Quoride uumalns Pi, post-infeclion; apo4op'tn Wupidett; G-inhibitor}   protein TCA . Iichloroacclic acid: TBS . Tris-bullcred saline; and Ihought couple PCR conipicr, Fo/tenay Gain (eiclion 1231 50656374
Ktn Ecr Frcnilm Frinkdin Glsal +aa Pcrgamon 0006.2952(94100294-0 EXPRESSION OF A CANNABINOID RECEPTOR IN BACULOVIRUS-INFECTED INSECT CELLS DEYISE DovE PETTIT , VINcFXT M. SHOwALTER MARY ABOOD Gur CAHRAL+ Depattncnts Microbiolocx(Immunalaz  and *Pharmacology Toxicologr , Medicul Collcgc Mineinia  Vrelu_A Commenucalh Univeesity, Richmond, VA 23298-0678. USA [Reciued Februars 1924; ecceptd Mey 1594) Abslract cannabinoid [eceplor recumbinani buculovins (AcNPV-THCR) has bcen conslrucied JIUJ emploted eiprceS0t ncun)l cnnn-cinoid Tccsciors Norzhern analysis ol [Olel RNAIrom Spodo Iera Acufiverud  Si Inxcl cclls inlecled with AcNPV THICR revcakd notr] hyPct-Pro Juctior of j)3kb uranschci Dinca probc [ mIcE-amslaled Mc cannabin vid [Cccpor cDNA Optimal viral niorcin cxaression "s cheetdd ~ntctabolically Iabeled AcVPV THCR-infecied St9 cclka multplacit" amaccnn n 2,5 , Tvensrrissior cleclrun Mictucunt cNFV THCR-infecicd Si9 ccl> showed cxicosl jucinGTMC per Lrbilion and ele ztron densi cMonlasmic Drtinucic? accumnulativn Indicatine cCcnio Zircontoicin cxniceion inmnunonuonescence suiminz using wnllctum prouuced (usicn ctolcir conskstinz cricinai damain Inc cannabinoid fcccpLor Jnd ncpzllis Anecm Ecueaucd cannabinoid Icccplor #IpTeS1CT AcNPy ~THCR-infcctcd Sf? cclls Scaichanur Rurnthil anihys ['HJCPSS , JJIU ICccptor birding indizaicd Kol JnMand j E4u11 emol/mg nqulcin Wuestern immunoblotting Deficlmcd AcNPV-THCR-inlected Stv ccll Iyeatcs rcvcaki Immunorc12042 band; Win rclativc molcculat #cights anging Iron ZokDa neeuonimam{ "ecics (55 KDa) exhibited 2 rclativc molccul #cial Consisle IL Kh thal predicted Jor th? Iranslational nroduzh obtaincd Icomi thc cannabinoid Tleenar cDNA coding Eenucncg Tanealun UWnE AcNPVTHCR MRNA also hickcd S5 kDa immunorcictive snecies; Tnte dala Indicile Ihal tkc birulotins cxpfesslon Ssicmi Is 4 Yoblc mcons cxdicssina aclalvcl lotgs quantitics cannabincid Feccniar Icomhinan prolci Wads Tadeulinand bindins; cannal inoid feceptor: nfalcin-couplco (cccpOr , Duculovm? crficssion ssicm; Celta-9-Ictrahydrocannabinol: amti canaabinuid receptor anliserum THC:, major psychoactive comconcn signal transduction pathways. stimulate inhibit miarijuana can produce multiplicity eflects In mcmoranc-associaico enzymes, andalter Tcmbranc numans including altcrations niood perccption . A 4ciaico chanucls . Howevct, stcrco cognition, memon psychomotor ac;ivity, Waa5 specilicity and KatCm Icqwtenienis or physio - gesia amliemesis,andimmiunosuppression [1 . 2]. logical activity suezest thal the cannabinoids also unlikely that onc mcchanism responsible cal a01 specilic receplor [6-10] the   multiplicity ccc onserieo uith THC Cannabinoid inducedinhibition ofcAMI' accumu- exposure Indeed, substanlial evidence exis'$ for two lation mouSC neuroblastoma ccll Jinc , NISTG2 , possible mcchanisms THC induced cellular Lelc"  lirs[delinilive evidence the presence effects. The highly lipophilic   nalure of THC has cunn pinoio [eceplots neunal esua [": suegested cmt ol  thc cannabinold-induced Radioligand binding studies that utilized potent ellects meMbrane perturbation [3-5] tritiarcd THC aniloz CFS5,910. utthz Such  disruption of cellular membranes may alter canbd of cannabinoid rcccplors in Rzjsence Ticun7 tissuC Antomical distnbution or the Corresponding aunor Dc; Guy Cacnal Mddicl cannabinoid Icceptor in rat brain #1$ characierized Wiieinij Aezini  Commonteill Univeri. [ccentor autoradiography using |HJCPSS,9410, Colleesints Box MCV Station Richmond VA 23298-0678, cicnsc binding observed in cerebellum 825-2306: FAX (5u4) 823-99+6. hppocampus basal ganglia [13,14 The Atorcsiations: TKC dalte-O-tetrehudrccannnbol: screndipitous discovery cDNA ckinc isolated AcNPV Amlofrplia calfforaica mullpi cnvclopcd troin ral cetehral corer cDNA library , shown nucet polyhedrosis ACNPV Gol We[oSals bind and  (unclionally Inicracr Cannalinoids recombininl bacuiovinus blocking tulict THCR, ahceseo fur the  miolecular chaclenizillion cannah naid feceptor; AcNPV-THCR naouolu (cccdl this receptor [s, 1S]. Sequencc analysis of this clone Iecombinant Oaculovirus GPCR Dfolci-Coup_cd Iercued Icceplor; HB-Ag.hcpatilis B core anligen: HBcAg THCR, high Jevel homolozy with GPCR hepattis 8 cofc onmeencannioinoidicccpronisonprolcim: tnar  contaimg erten? dnmain thal mesesses MOI , multiplicilg olinlecticn;PMSF . phenylm:tkylsulfoayl Icos aliun slcs even consen Lranememntane Quoride Pi, post-infeclion; apo4op'tn Wupidett; uumalns and an unletnal or cyloplasmic domain thal TCA . Iichloroacclic acid: TBS . Tris-bullcred saline; and Ihought couple G-inhibitor}   protein PCR Fo/tenay Gain (eiclion conipicr, 1231 50656374