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/ Bidirectional allosteric interactions between cannabinoid receptor 1 (CB1) and dopamine receptor 2 long (D2L) heterotetramers. Bidirectional allosteric interactions between cannabinoid receptor 1 (CB1) and dopamine receptor 2 long (D2L) heterotetramers. European journal of pharmacology 2017 imex curation 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 03-Oct-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation 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 Accepted 2019-SEP-25 AT 14:19 BST AT 14:19 BST by PPORRAS European journal of pharmacology 2017 Only protein-protein interactions imex curation imex curation Bidirectional allosteric interactions between cannabinoid receptor 1 (CB1) and dopamine receptor 2 long (D2L) heterotetramers. Bidirectional allosteric interactions between cannabinoid receptor 1 (CB1) and dopamine receptor 2 long (D2L) heterotetramers. European journal of pharmacology 2017 imex curation 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 03-Oct-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation 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 Accepted 2019-SEP-25 AT 14:19 BST AT 14:19 BST by PPORRAS European journal of pharmacology 2017 Only protein-protein interactions imex curation imex curation Bidirectional allosteric interactions between cannabinoid receptor 1 (CB1) and dopamine receptor 2 long (D2L) heterotetramers. Bidirectional allosteric interactions between cannabinoid receptor 1 (CB1) and dopamine receptor 2 long (D2L) heterotetramers. European journal of pharmacology 2017 imex curation 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 03-Oct-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation 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 Accepted 2019-SEP-25 AT 14:19 BST AT 14:19 BST by PPORRAS European journal of pharmacology 2017 Only protein-protein interactions imex curation imex curation Bidirectional allosteric interactions between cannabinoid receptor 1 (CB1) and dopamine receptor 2 long (D2L) heterotetramers. Bidirectional allosteric interactions between cannabinoid receptor 1 (CB1) and dopamine receptor 2 long (D2L) heterotetramers. European journal of pharmacology 2017 imex curation 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 03-Oct-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation 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. I have mostly given up on Supplementary fig 6: cannot make out the "key" as the colours/markings in the key and on the image do not correlate-there are no "grey" blocs; so have entered according Supp 6 results as best as I can deduce - I think Fig S6A interaction between B2 and GAi is negative (see Fig 3A). Also the main text states that in Supp. fig 6B- "we measured the effect of the co-expression and co-activation of D 2L and mGluR6 receptors on β-arrestin1 recruitment to D 2L (Suppl. Fig. 6B)". I do not see either b-arrestin nor mGluR6 in this figure. (they must mean Supp fig 8) 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 Accepted 2019-SEP-25 AT 14:19 BST AT 14:19 BST by PPORRAS European journal of pharmacology 2017 Only protein-protein interactions imex curation imex curation 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 CD541181FC4412EF arrb1_rat Beta-arrestin-1 Arrb1 Arrestin beta-1 protein protein rat Rattus norvegicus (Rat) Rat MGDKGTRVFKKASPNGKLTVYLGKRDFVDHIDLVDPVDGVVLVDPEYLKERRVYVTLTCAFRYGREDLDVLGLTFRKDLFVANVQSFPPAPEDKKPLTRLQERLIKKLGEHAYPFTFEIPPNLPCSVTLQPGPEDTGKACGVDYEVKAFCAENLEEKIHKRNSVRLVIRKVQYAPERPGPQPTAETTRQFLMSDKPLHLEASLDKEIYYHGEPISVNVHVTNNTNKTVKKIKISVRQYADICLFNTAQYKCPVAMEEADDTVAPSSTFCKVYTLTPFLANNREKRGLALDGKLKHEDTNLASSTLLREGANREILGIIVSYKVKVKLVVSRGGLLGDLASSDVAVELPFTLMHPKPKEEPPHREVPESETPVDTNLIELDTNDDDIVFEDFARQRLKGMKDDKDEEDDGTGSPHLNNR DIP protein P29066 original sequence version: 70 DIP protein P29066 original sequence version: 90 DIP protein P29066 original sequence version: 79 0A3C07D71B7ABC55 cnr1_human Cannabinoid receptor 1 CNR1 CNR CANN6 protein protein human Homo sapiens Human MKSILDGLADTTFRTITTDLLYVGSNDIQYEDIKGDMASKLGYFPQKFPLTSFRGSPFQEKMTAGDNPQLVPADQVNITEFYNKSLSSFKENEENIQCGENFMDIECFMVLNPSQQLAIAVLSLTLGTFTVLENLLVLCVILHSRSLRCRPSYHFIGSLAVADLLGSVIFVYSFIDFHVFHRKDSRNVFLFKLGGVTASFTASVGSLFLTAIDRYISIHRPLAYKRIVTRPKAVVAFCLMWTIAIVIAVLPLLGWNCEKLQSVCSDIFPHIDETYLMFWIGVTSVLLLFIVYAYMYILWKAHSHAVRMIQRGTQKSIIIHTSEDGKVQVTRPDQARMDIRLAKTLVLILVVLIICWGPLLAIMVYDVFGKMNKLIKTVFAFCSMLCLLNSTVNPIIYALRSKDLRHAFRSMFPSCEGTAQPLDNSMGDSDCLHKHANNAASVHRAAESCIKSTVKIAKVTMSVSTDTSAEAL DIP protein P21554 original sequence version: 162 1D2E49061D12ABF2 drd2_human D(2) dopamine receptor DRD2 Dopamine D2 receptor protein protein human Homo sapiens Human MDPLNLSWYDDDLERQNWSRPFNGSDGKADRPHYNYYATLLTLLIAVIVFGNVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMPWVVYLEVVGEWKFSRIHCDIFVTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMISIVWVLSFTISCPLLFGLNNADQNECIIANPAFVVYSSIVSFYVPFIVTLLVYIKIYIVLRRRRKRVNTKRSSRAFRAHLRAPLKGNCTHPEDMKLCTVIMKSNGSFPVNRRRVEAARRAQELEMEMLSSTSPPERTRYSPIPPSHHQLTLPDPSHHGLHSTPDSPAKPEKNGHAKDHPKIAKIFEIQTMPNGKTRTSLKTMSRRKLSQQKEKKATQMLAIVLGVFIICWLPFFITHILNIHCDCNIPPVLYSAFTWLGYVNSAVNPIIYTTFNIEFRKAFLKILHC 9BF8EA36C988A2E2 adrb2_human Beta-2 adrenergic receptor Beta-2 adrenoreceptor ADRB2 ADRB2R B2AR protein protein human Homo sapiens Human MGQPGNGSAFLLAPNGSHAPDHDVTQERDEVWVVGMGIVMSLIVLAIVFGNVLVITAIAKFERLQTVTNYFITSLACADLVMGLAVVPFGAAHILMKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAITSPFKYQSLLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINCYANETCCDFFTNQAYAIASSIVSFYVPLVIMVFVYSRVFQEAKRQLQKIDKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLKEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQDNLIRKEVYILLNWIGYVNSGFNPLIYCRSPDFRIAFQELLCLRRSSLKAYGNGYSSNGNTGEQSGYHVEQEKENKLLCEDLPGTEDFVGHQGTVPSDNIDSQGRNCSTNDSLL DIP protein P07550 original sequence version: 180 DIP protein P07550 original sequence version: 144 408C22731C6EDFBE 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 9F88311B46E62DE3 cnr1-cnr1-1 4 7 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 7 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 proximity proximity close-contact colocalization neighbourhood interaction Figure 1-1 here were no significant differences in BRET50 values among CB1homomers, D2Lhomomers and CB1/D2L heteromers. These findings demonstrated CB1and D2L receptors have similar affinities to form both homo and heteromers when expressed in HEK 293A cells. BRET results show that CB1and D2L receptors form both homo and heteromers, with no preference to form either. cnr1-cnr1-2 4 8 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 8 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 proximity proximity close-contact colocalization neighbourhood interaction Figure 1-2 BRET results show that CB1and D2L receptors form both homo and heteromers, with no preference to form either. cnr1-drd2-1 4 8 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 7 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 8 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 7 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 proximity proximity close-contact colocalization neighbourhood interaction 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-cnr1-3 1 7 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 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 7 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 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 physical association physical association Supplementary Figure 1 cnr1-cnr1-4 2 8 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 8 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 7 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 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 7 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 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 46 43 physical association physical association Figure 2, Supplementary Figure 3 drd2-cnr1-1 2 7 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 8 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 8 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 7 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 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 7 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 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 59 56 physical association physical association Figure 2C, Supplementary Figure 3 cnr1-gnai1-1 4 10 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 7 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 proximity proximity close-contact colocalization neighbourhood interaction 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 μM ACEA and increasing concentrations of the quinpirole resulted in concentration-dependent inhibition in BRET Eff between Gα i -Rluc and CB 1 -GFP 2 (Fig. 5B) 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 4 10 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 7 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 7 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 proximity proximity close-contact colocalization neighbourhood interaction 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 4 10 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 8 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 proximity proximity close-contact colocalization neighbourhood interaction 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. Fig 4-cannot add the peptide as a competitor here as CB1 is not detected- not an interactor. 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 4 9 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 5 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 proximity proximity close-contact colocalization neighbourhood interaction 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 4 5 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 7 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 proximity proximity close-contact colocalization neighbourhood interaction 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 4 5 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 8 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 proximity proximity close-contact colocalization neighbourhood interaction 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 4 6 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 7 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 proximity proximity close-contact colocalization neighbourhood interaction 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 4 6 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 8 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 proximity proximity close-contact colocalization neighbourhood interaction 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 4 6 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 7 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 7 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 proximity proximity close-contact colocalization neighbourhood interaction 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 4 6 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 7 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 8 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 proximity proximity close-contact colocalization neighbourhood interaction 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 4 10 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 7 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 7 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 proximity proximity close-contact colocalization neighbourhood interaction 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 4 10 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 8 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 proximity proximity close-contact colocalization neighbourhood interaction 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. added as a separate interaction as apart from the caution, the effects of blocking peptide plus ACEA AND Quinpirole are different for this figure and Figure 4 -see (EBI-21300793) cnr1-gnai1-3 3 10 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 7 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 proximity proximity close-contact colocalization neighbourhood interaction 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 3 5 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 7 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 proximity proximity close-contact colocalization neighbourhood interaction 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. arrb1-cnr1-3 4 6 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 7 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 proximity proximity close-contact colocalization neighbourhood interaction 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 4 6 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 8 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 proximity proximity close-contact colocalization neighbourhood interaction Supplementary figure 8B Untagged mGluR6 was co-expressed. Quinpirole (D2L agonist) concurrently with L-AP4 -an mGluR6 agonist, L-2-amino-4-phosphonobutyric acid. please check I have interpreted the "blobs" correctly as they are hard to see, and no 'correct' info in text for this figure. (stated to be Supp 6B) 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).