IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ http://www.ebi.ac.uk/ bagher-2017-1 human-293a Homo sapiens hek293a hek293a bret bioluminescence resonance energy transfer BRET LRET LRET BRET bret tag visualisation tag visualisation Bagher AM., Laprairie RB., Toguri JT., Kelly MEM., Denovan-Wright EM., Bagher AM., Laprairie RB., Toguri JT., Kelly MEM., Denovan-Wright EM., Bagher AM., Laprairie RB., Toguri JT., Kelly MEM., Denovan-Wright EM., Bagher AM., Laprairie RB., Toguri JT., Kelly MEM., Denovan-Wright EM. Two CB1 blocking peptides are used in this publication:- aas 300-431 and aas 432-456. As the 300-431 peptide specifically blocks the interaction between CB1 and GNAI1, the 432-456 (Khan and Lee, 2014) peptide has been assumed to have been used elsewhere. emdenova@dal.ca European journal of pharmacology 2017 Only protein-protein interactions imex curation imex curation bagher-2017-3 human-293a Homo sapiens hek293a hek293a fluorescence fluorescence technology fluorescence tag fluorescence tag visualisation by fluorescence tag fluorescence Bagher AM., Laprairie RB., Toguri JT., Kelly MEM., Denovan-Wright EM., Bagher AM., Laprairie RB., Toguri JT., Kelly MEM., Denovan-Wright EM., Bagher AM., Laprairie RB., Toguri JT., Kelly MEM., Denovan-Wright EM., Bagher AM., Laprairie RB., Toguri JT., Kelly MEM., Denovan-Wright EM. Sequential resonance energy transfer (SRET 2 ) combined with bimolecular complementation (BiFC). In this instance oxidation of the Rluc substrate coelenterazine by the donor Rluc-fused protein (D2L-Rluc) excites the BRET2 acceptor GFP2-fused protein (D2L-GFP2)and emission from GFP2 then excites the FRET acceptor -EYFP Venus (CB1 fused to the EYFP Venus N-terminal hemiprotein and CB1 fused to the EYFP Venus C hemiprotein). Two CB1 blocking peptides are used in this publication:- aas 300-431 and aas 432-456. As the 300-431 peptide specifically blocks the interaction between CB1 and GNAI1, the 432-456 (Khan and Lee, 2014) peptide has been assumed to have been used elsewhere. emdenova@dal.ca European journal of pharmacology 2017 Only protein-protein interactions imex curation imex curation bagher-2017-4 mouse-STHdhQ7/Q7 mouse STHdhQ7/Q7 sthdhq7/q7 STHdhQ7/Q7 mouse (brain)- striatal derived cell line from a knock in transgenic mouse containing homozygous Huntingtin (HTT) loci with a humanized Exon 1 containing 7 polyglutamine repeats. CVCL_M590 bret bioluminescence resonance energy transfer BRET LRET LRET BRET bret tag visualisation tag visualisation Bagher AM., Laprairie RB., Toguri JT., Kelly MEM., Denovan-Wright EM., Bagher AM., Laprairie RB., Toguri JT., Kelly MEM., Denovan-Wright EM., Bagher AM., Laprairie RB., Toguri JT., Kelly MEM., Denovan-Wright EM., Bagher AM., Laprairie RB., Toguri JT., Kelly MEM., Denovan-Wright EM. Two CB1 blocking peptides are used in this publication:- aas 300-431 and aas 432-456. As the 300-431 peptide specifically blocks the interaction between CB1 and GNAI1, the 432-456 (Khan and Lee, 2014) peptide has been assumed to have been used elsewhere. emdenova@dal.ca European journal of pharmacology 2017 Only protein-protein interactions imex curation imex curation bagher-2017-2 human-293a Homo sapiens hek293a hek293a bifc bimolecular fluorescence complementation bifc tag fluorescence tag visualisation by fluorescence tag fluorescence Bagher AM., Laprairie RB., Toguri JT., Kelly MEM., Denovan-Wright EM., Bagher AM., Laprairie RB., Toguri JT., Kelly MEM., Denovan-Wright EM., Bagher AM., Laprairie RB., Toguri JT., Kelly MEM., Denovan-Wright EM., Bagher AM., Laprairie RB., Toguri JT., Kelly MEM., Denovan-Wright EM. The Beta2 adrenergic receptor homodimerisation as shown in Supplementary figure 1B is not shown as constructs used cannot be traced from the stated reference;Hamad and Dupre 2010 (PMID:20868491). Two CB1 blocking peptides are used in this publication:- aas 300-431 and aas 432-456. As the 300-431 peptide specifically blocks the interaction between CB1 and GNAI1, the 432-456 (Khan and Lee, 2014) peptide has been assumed to have been used elsewhere. emdenova@dal.ca European journal of pharmacology 2017 Only protein-protein interactions imex curation imex curation cnr1_human Cannabinoid receptor 1 CNR1 CNR CANN6 protein protein human Homo sapiens Human MKSILDGLADTTFRTITTDLLYVGSNDIQYEDIKGDMASKLGYFPQKFPLTSFRGSPFQEKMTAGDNPQLVPADQVNITEFYNKSLSSFKENEENIQCGENFMDIECFMVLNPSQQLAIAVLSLTLGTFTVLENLLVLCVILHSRSLRCRPSYHFIGSLAVADLLGSVIFVYSFIDFHVFHRKDSRNVFLFKLGGVTASFTASVGSLFLTAIDRYISIHRPLAYKRIVTRPKAVVAFCLMWTIAIVIAVLPLLGWNCEKLQSVCSDIFPHIDETYLMFWIGVTSVLLLFIVYAYMYILWKAHSHAVRMIQRGTQKSIIIHTSEDGKVQVTRPDQARMDIRLAKTLVLILVVLIICWGPLLAIMVYDVFGKMNKLIKTVFAFCSMLCLLNSTVNPIIYALRSKDLRHAFRSMFPSCEGTAQPLDNSMGDSDCLHKHANNAASVHRAAESCIKSTVKIAKVTMSVSTDTSAEAL drd2_human D(2) dopamine receptor DRD2 Dopamine D2 receptor protein protein human Homo sapiens Human MDPLNLSWYDDDLERQNWSRPFNGSDGKADRPHYNYYATLLTLLIAVIVFGNVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMPWVVYLEVVGEWKFSRIHCDIFVTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMISIVWVLSFTISCPLLFGLNNADQNECIIANPAFVVYSSIVSFYVPFIVTLLVYIKIYIVLRRRRKRVNTKRSSRAFRAHLRAPLKGNCTHPEDMKLCTVIMKSNGSFPVNRRRVEAARRAQELEMEMLSSTSPPERTRYSPIPPSHHQLTLPDPSHHGLHSTPDSPAKPEKNGHAKDHPKIAKIFEIQTMPNGKTRTSLKTMSRRKLSQQKEKKATQMLAIVLGVFIICWLPFFITHILNIHCDCNIPPVLYSAFTWLGYVNSAVNPIIYTTFNIEFRKAFLKILHC gnai1_human Guanine nucleotide-binding protein G(i) subunit alpha-1 GNAI1 Adenylate cyclase-inhibiting G alpha protein protein protein human Homo sapiens Human MGCTLSAEDKAAVERSKMIDRNLREDGEKAAREVKLLLLGAGESGKSTIVKQMKIIHEAGYSEEECKQYKAVVYSNTIQSIIAIIRAMGRLKIDFGDSARADDARQLFVLAGAAEEGFMTAELAGVIKRLWKDSGVQACFNRSREYQLNDSAAYYLNDLDRIAQPNYIPTQQDVLRTRVKTTGIVETHFTFKDLHFKMFDVGGQRSERKKWIHCFEGVTAIIFCVALSDYDLVLAEDEEMNRMHESMKLFDSICNNKWFTDTSIILFLNKKDLFEEKIKKSPLTICYPEYAGSNTYEEAAAYIQCQFEDLNKRKDTKEIYTHFTCATDTKNVQFVFDAVTDVIIKNNLKDCGLF adrb2_human Beta-2 adrenergic receptor Beta-2 adrenoreceptor ADRB2 ADRB2R B2AR protein protein human Homo sapiens Human MGQPGNGSAFLLAPNGSHAPDHDVTQERDEVWVVGMGIVMSLIVLAIVFGNVLVITAIAKFERLQTVTNYFITSLACADLVMGLAVVPFGAAHILMKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAITSPFKYQSLLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINCYANETCCDFFTNQAYAIASSIVSFYVPLVIMVFVYSRVFQEAKRQLQKIDKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLKEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQDNLIRKEVYILLNWIGYVNSGFNPLIYCRSPDFRIAFQELLCLRRSSLKAYGNGYSSNGNTGEQSGYHVEQEKENKLLCEDLPGTEDFVGHQGTVPSDNIDSQGRNCSTNDSLL p63092-1 Guanine nucleotide-binding protein G(s) subunit alpha isoforms short Adenylate cyclase-stimulating G alpha protein GNAS GNAS1 GSP Alpha-S2 GNASl Alpha-S-long protein protein human Homo sapiens Human MGCLGNSKTEDQRNEEKAQREANKKIEKQLQKDKQVYRATHRLLLLGAGESGKSTIVKQMRILHVNGFNGEGGEEDPQAARSNSDGEKATKVQDIKNNLKEAIETIVAAMSNLVPPVELANPENQFRVDYILSVMNVPDFDFPPEFYEHAKALWEDEGVRACYERSNEYQLIDCAQYFLDKIDVIKQADYVPSDQDLLRCRVLTSGIFETKFQVDKVNFHMFDVGGQRDERRKWIQCFNDVTAIIFVVASSSYNMVIREDNQTNRLQEALNLFKSIWNNRWLRTISVILFLNKQDLLAEKVLAGKSKIEDYFPEFARYTTPEDATPEPGEDPRVTRAKYFIRDEFLRISTASGDGRHYCYPHFTCAVDTENIRRVFNDCRDIIQRMHLRQYELL arrb1_rat Beta-arrestin-1 Arrb1 Arrestin beta-1 protein protein rat Rattus norvegicus (Rat) Rat MGDKGTRVFKKASPNGKLTVYLGKRDFVDHIDLVDPVDGVVLVDPEYLKERRVYVTLTCAFRYGREDLDVLGLTFRKDLFVANVQSFPPAPEDKKPLTRLQERLIKKLGEHAYPFTFEIPPNLPCSVTLQPGPEDTGKACGVDYEVKAFCAENLEEKIHKRNSVRLVIRKVQYAPERPGPQPTAETTRQFLMSDKPLHLEASLDKEIYYHGEPISVNVHVTNNTNKTVKKIKISVRQYADICLFNTAQYKCPVAMEEADDTVAPSSTFCKVYTLTPFLANNREKRGLALDGKLKHEDTNLASSTLLREGANREILGIIVSYKVKVKLVVSRGGLLGDLASSDVAVELPFTLMHPKPKEEPPHREVPESETPVDTNLIELDTNDDDIVFEDFARQRLKGMKDDKDEEDDGTGSPHLNNR cnr1-cnr1-1 3556595 N/A 3556599 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed c-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false Stoichiometry: 1.0 N/A 3556599 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed c-terminus renilla luciferase renilla luciferase protein tag renilla luciferase c-term range C-terminal range c-term range c-term range C-terminal range c-term range false Stoichiometry: 1.0 proximity proximity close-contact colocalization neighbourhood interaction false false false Figure 1-1 BRET results show that CB1and D2L receptors form both homo and heteromers, with no preference to form either. cnr1-cnr1-2 3556595 N/A 3556600 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed c-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false Stoichiometry: 1.0 N/A 3556600 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed c-terminus renilla luciferase renilla luciferase protein tag renilla luciferase c-term range C-terminal range c-term range c-term range C-terminal range c-term range false Stoichiometry: 1.0 proximity proximity close-contact colocalization neighbourhood interaction false false false Figure 1-2 BRET results show that CB1and D2L receptors form both homo and heteromers, with no preference to form either. cnr1-drd2-1 3556595 N/A 3556600 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed c-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false N/A 3556599 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed c-terminus renilla luciferase renilla luciferase protein tag renilla luciferase c-term range C-terminal range c-term range c-term range C-terminal range c-term range false N/A 3556600 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed c-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false N/A 3556599 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed c-terminus renilla luciferase renilla luciferase protein tag renilla luciferase c-term range C-terminal range c-term range c-term range C-terminal range c-term range false proximity proximity close-contact colocalization neighbourhood interaction false false false Figure 1-3 BRET results show that CB1and D2L receptors form both homo and heteromers, with no preference to form either. Co-expression of CB1 and D2L resulted in a calculated oligomerization state value of N= 2.61 ± 0.24 (Fig. 1B), which implied that CB 1 /D 2L heteromers were present as tetramers or higher-order oligomeric complexes. cnr1-gnai1-1 3556595 N/A 3556601 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed Iso93-Asp94 renilla luciferase renilla luciferase protein tag renilla luciferase certain certain sequence position certain certain certain sequence position certain false N/A 3556599 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed c-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false proximity proximity close-contact colocalization neighbourhood interaction false false false Figure 3A Arachidonyl-2'-chloroethylamide (ACEA) - a selective, synthetic agonist of CB1R. Untagged D2L was co-expressed. Quinpirole (D2L agonist) added concurrently with Arachidonyl-2'-chloroethylamide (ACEA) - a selective, synthetic agonist of CB1R. Treating the cells with 1 uM ACEA and increasing the concentration of quinpirole resulted in concentration-dependent inhibition in BRET Eff between Gai-Rluc and CB1-GFP 2. Co-expression and co-activation of either D2L and beta2 did not alter CB1 coupling to G proteins. Quinpirole (D2L agonist) but only in the presence of untagged D2L. This increase in BRET Eff was PTx-sensitive. PTx (Pertussis toxin) blocked ACEA-dependent and also Quinpirole+D2L dependent increases in BRET Efficiency. cnr1-gnai1-2 3556595 N/A 3556601 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed Iso93-Asp94 renilla luciferase renilla luciferase protein tag renilla luciferase certain certain sequence position certain certain certain sequence position certain false N/A 3556599 competitor competitor competitor unspecified role unspecified role over-expressed over expressed level over-expressed region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false N/A 3556599 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed c-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false proximity proximity close-contact colocalization neighbourhood interaction false false false Figure 4B-2 Arachidonyl-2'-chloroethylamide (ACEA) - a selective, synthetic agonist of CB1R. In the presence of the blocking peptide agonist activity was reduced. Untagged D2L was co-expressed. Quinpirole (D2L agonist). The presence of the blocking peptide did not significantly affect the action of Quinpirole. cnr1-cnr1-5 3556595 N/A 3556601 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed Iso93-Asp94 renilla luciferase renilla luciferase protein tag renilla luciferase certain certain sequence position certain certain certain sequence position certain false N/A 3556600 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed c-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false proximity proximity close-contact colocalization neighbourhood interaction false false false Figure 3C, 4D, 5E/F, Supplementary fig 2C, 5B,6B Arachidonyl-2'-chloroethylamide (ACEA) - a selective, synthetic agonist of CB1R. In the presence of the CB1 blocking peptide (aas 300-431) agonist activity was reduced. Untagged CB1 was co-expressed. Quinpirole (D2L agonist). The presence of the blocking peptide did not significantly affect the action of Quinpirole. A reduction in BRET Eff signals between Gai-Rluc and D2L-GFP 2 was observed in cells treated with 1 uM quinpirole and increasing concentrations of ACEA (Fig. 5E). Quinpirole (D2L agonist) added concurrently with Arachidonyl-2'-chloroethylamide (ACEA) - a selective, synthetic agonist of CB1R. Co-expression and co-activation of either CB1 and beta2 did not alter D2 coupling to G proteins. PTx (Pertussis toxin) blocked Quinpirole-dependent and also ACEA+CB1 dependent increases in BRET Efficiency. drd2-gnas-1 3556595 N/A 3556602 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 3556603 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed Ala188-Asp189 renilla luciferase renilla luciferase protein tag renilla luciferase certain certain sequence position certain certain certain sequence position certain false proximity proximity close-contact colocalization neighbourhood interaction false false false Supplementary Fig 4A/B +6B/D isoprenaline - a Beta2 agonist Cholera toxin (CTx) Co-expression and co-activation of CB1 did not alter coupling to G proteins. adrb2-gnas-5 3556595 N/A 3556603 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed Ala188-Asp189 renilla luciferase renilla luciferase protein tag renilla luciferase certain certain sequence position certain certain certain sequence position certain false N/A 3556599 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed C-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false proximity proximity close-contact colocalization neighbourhood interaction false false false Fig 6B/C, Suppl. Fig. 2B,6B Untagged D2L was co-expressed. Co-application of ACEA and quinpirole. Application of either compound alone had marginal effect. Co-expression and co-activation of either D2L and beta2 did not alter CB1 or D2 coupling to G proteins. cnr1-gnas-1 3556595 N/A 3556603 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed Ala188-Asp189 renilla luciferase renilla luciferase protein tag renilla luciferase certain certain sequence position certain certain certain sequence position certain false N/A 3556600 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed C-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false proximity proximity close-contact colocalization neighbourhood interaction false false false Fig 6E/F , Suppl. Fig. 2D, 6D Untagged CB1 was co-expressed. Co-application of ACEA and quinpirole. Application of either compound alone had marginal effect. Co-expression and co-activation of either CB1 and beta2 did not alter D2 coupling to G proteins. cnr1-cnr1-6 3556595 N/A 3556604 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed C-terminus renilla luciferase renilla luciferase protein tag renilla luciferase c-term range C-terminal range c-term range c-term range C-terminal range c-term range false N/A 3556599 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed c-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false proximity proximity close-contact colocalization neighbourhood interaction false false false Figure 9, 11B. Arachidonyl-2'-chloroethylamide (ACEA) - a selective, synthetic agonist of CB1R. Untagged D2L was co-expressed. Quinpirole (D2L agonist). Co-application of both ACEA and quinpirole significantly potentiated BRET Eff signal between Beta-arrestin1-Rluc and CB 1-GFP compared to ACEA-treated cells. arrb1-cnr1-1 3556595 N/A 3556604 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed C-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false N/A 3556600 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed c-terminus renilla luciferase renilla luciferase protein tag renilla luciferase c-term range C-terminal range c-term range c-term range C-terminal range c-term range false proximity proximity close-contact colocalization neighbourhood interaction false false false Figure 10, 11D, Supplementary Figure 8B Arachidonyl-2'-chloroethylamide (ACEA) - a selective, synthetic agonist of CB1R. Untagged CB1 was co-expressed. Quinpirole (D2L agonist). Co-application of both ACEA and quinpirole significantly increased BRET Eff signal as compared to BRET Eff signals obtained from cells treated with each agonist individually. arrb1-cnr1-2 3556595 N/A 3556604 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed C-terminus renilla luciferase renilla luciferase protein tag renilla luciferase c-term range C-terminal range c-term range c-term range C-terminal range c-term range false N/A 3556599 competitor competitor competitor unspecified role unspecified role over-expressed over expressed level over-expressed region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false N/A 3556599 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed c-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false proximity proximity close-contact colocalization neighbourhood interaction false false false Figure 11B Arachidonyl-2'-chloroethylamide (ACEA) - a selective, synthetic agonist of CB1R. Untagged D2L was co-expressed. Quinpirole (D2L agonist). In cells expressing CB1-BP lower energy transfer was observed between cells treated with quinpirole or co-treated with quinpirole and ACEA, as compared to cells treated with the same agonist(s) expressing an empty pcDNA. No change in the energy transfer was observed in cells expressing CB1-BP following ACEA treatment compared to cells transfected with empty pcDNA. arrb1-drd2-1 3556595 N/A 3556604 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed C-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false N/A 3556599 competitor competitor competitor unspecified role unspecified role over-expressed over expressed level over-expressed region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false N/A 3556600 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed c-terminus renilla luciferase renilla luciferase protein tag renilla luciferase c-term range C-terminal range c-term range c-term range C-terminal range c-term range false proximity proximity close-contact colocalization neighbourhood interaction false false false Figure 11D Arachidonyl-2'-chloroethylamide (ACEA) - a selective, synthetic agonist of CB1R. Untagged CB1 was co-expressed. Quinpirole (D2L agonist). Co-expression of CB1-BP reduced the BRET signal in cells treated with ACEA or ACEA plus Quinpirole. Treating the cells with quinpirole alone did not alter BRET signal in cells expressing CB1-BP compared to those expressing empty pcDNA. cnr1-cnr1-7 3556595 N/A 3556601 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed Iso93-Asp94 renilla luciferase renilla luciferase protein tag renilla luciferase certain certain sequence position certain certain certain sequence position certain false N/A 3556599 competitor competitor competitor unspecified role unspecified role over-expressed over expressed level over-expressed region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false N/A 3556599 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed c-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false proximity proximity close-contact colocalization neighbourhood interaction false false false Supplementary Figure 5A Arachidonyl-2'-chloroethylamide (ACEA) - a selective, synthetic agonist of CB1R. In the presence of the blocking peptide agonist activity was not affected. Untagged D2L was co-expressed. Quinpirole (D2L agonist). The presence of the blocking peptide reduced the activity of Quinpirole. The co-expression of CB1/D2L hetero-oligomer blocking peptide (CB1-BP), which inhibited the physical interaction between CB1 and D2 (Fig. 2C), inhibited the switch of CB1 and D2L coupling from Ga i to Gas proteins following co-activation of both receptors (Suppl. Fig. 5A,B). ACEA plus Quinpirole (D2L agonist):-in the presence of CB1 blocking peptide the BRET signal increased. cnr1-cnr1-8 3556595 N/A 3556601 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed Iso93-Asp94 renilla luciferase renilla luciferase protein tag renilla luciferase certain certain sequence position certain certain certain sequence position certain false N/A 3556600 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed c-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false proximity proximity close-contact colocalization neighbourhood interaction false false false Supplementary Figure 5B Arachidonyl-2'-chloroethylamide (ACEA) - a selective, synthetic agonist of CB1R. In the presence of the blocking peptide agonist activity was decreased. Untagged CB1 was co-expressed. Quinpirole (D2L agonist). The presence of the blocking peptide did not affect the activity of Quinpirole. The co-expression of CB1/D2L hetero-oligomer blocking peptide (CB1-BP aas 432-456), which inhibited the physical interaction between CB1 and D2 (Fig. 2C), inhibited the switch of CB1 and D2L coupling from Ga i to Gas proteins following co-activation of both receptors (Suppl. Fig. 5A,B). ACEA plus Quinpirole (D2L agonist):-in the presence of CB1 blocking peptide the BRET signal increased. The figure legend and figure text give different interactors for this figure- the interactors from the figure legend have been assumed to be the correct ones. arrb1-cnr1-3 3556595 N/A 3556604 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed C-terminus renilla luciferase renilla luciferase protein tag renilla luciferase c-term range C-terminal range c-term range c-term range C-terminal range c-term range false N/A 3556599 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed c-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false proximity proximity close-contact colocalization neighbourhood interaction false false false Suppl. Fig. 8A Arachidonyl-2'-chloroethylamide (ACEA) - a selective, synthetic agonist of CB1R. Untagged mGluR6 was co-expressed. mGluR6 agonist L-2-amino-4-phosphonobutyric acid (L-AP4) co-application of both ACEA and 1 L-AP4 resulted in similar BRET Eff signal between b-arrestin1-Rluc and CB1-GFP 2 as compared to cells treated with ACEA only. arrb1-drd2-2 3556595 N/A 3556604 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed C-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false N/A 3556600 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed c-terminus renilla luciferase renilla luciferase protein tag renilla luciferase c-term range C-terminal range c-term range c-term range C-terminal range c-term range false proximity proximity close-contact colocalization neighbourhood interaction false false false Supplementary figure 8B Untagged mGluR6 was co-expressed. Quinpirole (D2L agonist) concurrently with L-AP4 -an mGluR6 agonist, L-2-amino-4-phosphonobutyric acid. The co-expression of un-tagged mGluR6 together with D 2L -Rluc and b-arrestin1-GFP 2 did not alter quinpirole-induced b-arrestin1 recruitment to D2L receptors in the absence or presence of the mGluR6 agonist (L-AP4). cnr1-cnr1-4 3556596 N/A 3556600 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed c-terminus renilla luciferase renilla luciferase protein tag renilla luciferase c-term range C-terminal range c-term range c-term range C-terminal range c-term range false N/A 3556600 unspecified role unspecified role luminescence transmitter luminescence transmitter over-expressed over expressed level over-expressed c-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false N/A 3556599 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept over-expressed over expressed level over-expressed c-terminus venus-n venus n-terminal fragment c-term range C-terminal range c-term range c-term range C-terminal range c-term range false n-terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false N/A 3556599 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept over-expressed over expressed level over-expressed c-terminus venus-c venus c-terminal fragment c-term range C-terminal range c-term range c-term range C-terminal range c-term range false n-terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false 3556711 3556708 physical association physical association false false false Figure 2, Supplementary Figure 3 drd2-cnr1-1 3556596 N/A 3556599 competitor competitor competitor neutral component neutral component over-expressed over expressed level over-expressed region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false N/A 3556600 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed c-terminus renilla luciferase renilla luciferase protein tag renilla luciferase c-term range C-terminal range c-term range c-term range C-terminal range c-term range false N/A 3556600 unspecified role unspecified role luminescence transmitter luminescence transmitter over-expressed over expressed level over-expressed c-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false N/A 3556599 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept over-expressed over expressed level over-expressed c-terminus venus-n venus n-terminal fragment c-term range C-terminal range c-term range c-term range C-terminal range c-term range false n-terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false N/A 3556599 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept over-expressed over expressed level over-expressed c-terminus venus-c venus c-terminal fragment c-term range C-terminal range c-term range c-term range C-terminal range c-term range false n-terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false 3556724 3556721 physical association physical association false false false Figure 2C, Supplementary Figure 3 cnr1-gnai1-3 3556597 N/A 3556601 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed Iso93-Asp94 renilla luciferase renilla luciferase protein tag renilla luciferase certain certain sequence position certain certain certain sequence position certain false N/A 3556599 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed c-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false proximity proximity close-contact colocalization neighbourhood interaction false false false Supplementary Fig 7A Arachidonyl-2'-chloroethylamide (ACEA) - a selective, synthetic agonist of CB1R. Quinpirole (D2L agonist) added concurrently with Arachidonyl-2'-chloroethylamide (ACEA) (a selective, synthetic agonist of CB1R), but only when untagged D2L was co-expressed. This effect was reduced by addition of O-2050 -a small molecule cannabinoid derivative, acting as an antagonist for the CB1 receptor. cnr1-gnas-2 3556597 N/A 3556603 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed Ala188-Asp189 renilla luciferase renilla luciferase protein tag renilla luciferase certain certain sequence position certain certain certain sequence position certain false N/A 3556599 unspecified role unspecified role luminescence accept luminescence acceptor luminescence accept over-expressed over expressed level over-expressed c-terminus gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false proximity proximity close-contact colocalization neighbourhood interaction false false false Supplementary Fig 7B Quinpirole (D2L agonist) added concurrently with Arachidonyl-2'-chloroethylamide (ACEA) (a selective, synthetic agonist of CB1R), but only when untagged D2L was co-expressed. This effect was eradicated by addition of O-2050 -a small molecule cannabinoid derivative, acting as an antagonist for the CB1 receptor. cnr1-cnr1-3 3556598 N/A 3556599 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed c-terminus venus-n venus n-terminal fragment c-term range C-terminal range c-term range c-term range C-terminal range c-term range false n-terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false N/A 3556599 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed c-terminus venus-c venus c-terminal fragment c-term range C-terminal range c-term range c-term range C-terminal range c-term range false n-terminus ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false physical association physical association false false false Supplementary Figure 1