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/ The M(5) muscarinic acetylcholine receptor third intracellular loop regulates receptor function and oligomerization. The M(5) muscarinic acetylcholine receptor third intracellular loop regulates receptor function and oligomerization. Biochimica et biophysica acta 2010 imex curation Borroto-Escuela DO., García-Negredo G., Garriga P., Fuxe K., Ciruela F. fciruela@ub.edu 25-Jul-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. bret bioluminescence resonance energy transfer BRET LRET LRET BRET bret tag visualisation tag visualisation Borroto-Escuela DO., García-Negredo G., Garriga P., Fuxe K., Ciruela F. fciruela@ub.edu Accepted 2019-JUN-24 AT 16:17 BST AT 16:17 BST by LIVIAP Biochimica et biophysica acta 2010 Only protein-protein interactions imex curation imex curation altered; altered The M(5) muscarinic acetylcholine receptor third intracellular loop regulates receptor function and oligomerization. The M(5) muscarinic acetylcholine receptor third intracellular loop regulates receptor function and oligomerization. Biochimica et biophysica acta 2010 imex curation Borroto-Escuela DO., García-Negredo G., Garriga P., Fuxe K., Ciruela F. fciruela@ub.edu 25-Jul-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. confocal microscopy confocal microscopy immunocytochemistry immunocytochemistry Borroto-Escuela DO., García-Negredo G., Garriga P., Fuxe K., Ciruela F. fciruela@ub.edu Accepted 2019-JUN-24 AT 16:17 BST AT 16:17 BST by LIVIAP Biochimica et biophysica acta 2010 Only protein-protein interactions imex curation imex curation altered; altered The M(5) muscarinic acetylcholine receptor third intracellular loop regulates receptor function and oligomerization. The M(5) muscarinic acetylcholine receptor third intracellular loop regulates receptor function and oligomerization. Biochimica et biophysica acta 2010 imex curation Borroto-Escuela DO., García-Negredo G., Garriga P., Fuxe K., Ciruela F. fciruela@ub.edu 25-Jul-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti bait coip anti bait coimmunoprecipitation anti bait coip anti tag western anti tag western blot anti tag western Borroto-Escuela DO., García-Negredo G., Garriga P., Fuxe K., Ciruela F. fciruela@ub.edu Accepted 2019-JUN-24 AT 16:17 BST AT 16:17 BST by LIVIAP Biochimica et biophysica acta 2010 Only protein-protein interactions imex curation imex curation altered; altered The M(5) muscarinic acetylcholine receptor third intracellular loop regulates receptor function and oligomerization. The M(5) muscarinic acetylcholine receptor third intracellular loop regulates receptor function and oligomerization. Biochimica et biophysica acta 2010 imex curation Borroto-Escuela DO., García-Negredo G., Garriga P., Fuxe K., Ciruela F. fciruela@ub.edu 25-Jul-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-molt-3 MOLT-3 Adult T acute lymphoblastic leukemia molt-3 anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Borroto-Escuela DO., García-Negredo G., Garriga P., Fuxe K., Ciruela F. fciruela@ub.edu Accepted 2019-JUN-24 AT 16:17 BST AT 16:17 BST by LIVIAP Biochimica et biophysica acta 2010 Only protein-protein interactions imex curation imex curation altered; altered The M(5) muscarinic acetylcholine receptor third intracellular loop regulates receptor function and oligomerization. The M(5) muscarinic acetylcholine receptor third intracellular loop regulates receptor function and oligomerization. Biochimica et biophysica acta 2010 imex curation Borroto-Escuela DO., García-Negredo G., Garriga P., Fuxe K., Ciruela F. fciruela@ub.edu 25-Jul-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Borroto-Escuela DO., García-Negredo G., Garriga P., Fuxe K., Ciruela F. fciruela@ub.edu Accepted 2019-JUN-24 AT 16:17 BST AT 16:17 BST by LIVIAP Biochimica et biophysica acta 2010 Only protein-protein interactions imex curation imex curation altered; altered The M(5) muscarinic acetylcholine receptor third intracellular loop regulates receptor function and oligomerization. The M(5) muscarinic acetylcholine receptor third intracellular loop regulates receptor function and oligomerization. Biochimica et biophysica acta 2010 imex curation Borroto-Escuela DO., García-Negredo G., Garriga P., Fuxe K., Ciruela F. fciruela@ub.edu 25-Jul-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-molt-3 MOLT-3 Adult T acute lymphoblastic leukemia molt-3 confocal microscopy confocal microscopy immunocytochemistry immunocytochemistry Borroto-Escuela DO., García-Negredo G., Garriga P., Fuxe K., Ciruela F. fciruela@ub.edu Accepted 2019-JUN-24 AT 16:17 BST AT 16:17 BST by LIVIAP Biochimica et biophysica acta 2010 Only protein-protein interactions imex curation imex curation altered; altered acm3_human Muscarinic acetylcholine receptor M3 CHRM3 protein protein human Homo sapiens Human MTLHNNSTTSPLFPNISSSWIHSPSDAGLPPGTVTHFGSYNVSRAAGNFSSPDGTTDDPLGGHTVWQVVFIAFLTGILALVTIIGNILVIVSFKVNKQLKTVNNYFLLSLACADLIIGVISMNLFTTYIIMNRWALGNLACDLWLAIDYVASNASVMNLLVISFDRYFSITRPLTYRAKRTTKRAGVMIGLAWVISFVLWAPAILFWQYFVGKRTVPPGECFIQFLSEPTITFGTAIAAFYMPVTIMTILYWRIYKETEKRTKELAGLQASGTEAETENFVHPTGSSRSCSSYELQQQSMKRSNRRKYGRCHFWFTTKSWKPSSEQMDQDHSSSDSWNNNDAAASLENSASSDEEDIGSETRAIYSIVLKLPGHSTILNSTKLPSSDNLQVPEEELGMVDLERKADKLQAQKSVDDGGSFPKSFSKLPIQLESAVDTAKTSDVNSSVGKSTATLPLSFKEATLAKRFALKTRSQITKRKRMSLVKEKKAAQTLSAILLAFIITWTPYNIMVLVNTFCDSCIPKTFWNLGYWLCYINSTVNPVCYALCNKTFRTTFKMLLLCQCDKKKRRKQQYQQRQSVIFHKRAPEQAL DIP protein P20309 original sequence version: 117 5CB473C57B9526E9 acm5_human Muscarinic acetylcholine receptor M5 CHRM5 protein protein human Homo sapiens Human MEGDSYHNATTVNGTPVNHQPLERHRLWEVITIAAVTAVVSLITIVGNVLVMISFKVNSQLKTVNNYYLLSLACADLIIGIFSMNLYTTYILMGRWALGSLACDLWLALDYVASNASVMNLLVISFDRYFSITRPLTYRAKRTPKRAGIMIGLAWLISFILWAPAILCWQYLVGKRTVPLDECQIQFLSEPTITFGTAIAAFYIPVSVMTILYCRIYRETEKRTKDLADLQGSDSVTKAEKRKPAHRALFRSCLRCPRPTLAQRERNQASWSSSRRSTSTTGKPSQATGPSANWAKAEQLTTCSSYPSSEDEDKPATDPVLQVVYKSQGKESPGEEFSAEETEETFVKAETEKSDYDTPNYLLSPAAAHRPKSQKCVAYKFRLVVKADGNQETNNGCHKVKIMPCPFPVAKEPSTKGLNPNPSHQMTKRKRVVLVKERKAAQTLSAILLAFIITWTPYNIMVLVSTFCDKCVPVTLWHLGYWLCYVNSTVNPICYALCNRTFRKTFKMLLLCRWKKKKVEEKLYWQGNSKLP EE846186F645DD9B chrm3-chrm5-1 5 7 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed 8 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 2 chrm3-chrm5-2 2 7 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization Figure 1B chrm5-chrm3-1 3 7 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed 8 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 2 chrm3-chrm5-3 1 7 unspecified role unspecified role fluorescence donor fluorescence 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 fluorescence accept fluorescence acceptor fluorescence 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/B chrm3-chrm5-4 1 7 unspecified role unspecified role fluorescence donor fluorescence 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 fluorescence accept fluorescence acceptor fluorescence 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 1 chrm3-chrm5-5 1 8 unspecified role unspecified role fluorescence donor fluorescence 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 fluorescence accept fluorescence acceptor fluorescence 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 2 chrm3-chrm5-6 1 8 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence 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 fluorescence donor fluorescence 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 competitor competitor competitor unspecified role unspecified role over-expressed over expressed level over-expressed n-terminus sbp sbp n-term range n-terminal range n-term range n-term range n-terminal range n-term range n-terminus cam tag calmodulin binding protein tag cam tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range cytoplasmic_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain proximity proximity close-contact colocalization neighbourhood interaction Figure 3B Expression of M5R-235–409 peptide promoted a 30% reduction in the BRETmax, with no significant change in the BRET50. from looking at Fig 4A I would think the construct does have the N-terminal tags present, although these are not mentioned elsewhere for Fig 3B. However I cannot confirm this. chrm3-chrm5-7 2 8 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed n-terminus cam tag calmodulin binding protein tag cam tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range n-terminus sbp sbp n-term range n-terminal range n-term range n-term range n-terminal range n-term range cytoplasmic_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 neutral component neutral component over-expressed over expressed level over-expressed region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization Figure 5B M3R was also cotransfected. chrm3-chrm5-8 6 7 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level 8 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level colocalization colocalization Figure 8A chrm5-chrm3-2 4 7 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level 8 unspecified role unspecified role bait bait physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association Figure 8B lane 3(4)