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/ Human TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and trafficking. Human TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and trafficking. J. Biol. Chem. (0021-9258) 2006 imex curation Arniges M., Fernandez-Fernandez JM., Albrecht N., Schaefer M., Valverde MA. BioCreative - Critical Assessment of Information Extraction systems in Biology miguel.valverde@upf.edu 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 confocal microscopy confocal microscopy antibody detection identification by antibody antibody detection Arniges M., Fernandez-Fernandez JM., Albrecht N., Schaefer M., Valverde MA. BioCreative - Critical Assessment of Information Extraction systems in Biology miguel.valverde@upf.edu Accepted 2006-AUG-4 by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation Human TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and trafficking. Human TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and trafficking. J. Biol. Chem. (0021-9258) 2006 imex curation Arniges M., Fernandez-Fernandez JM., Albrecht N., Schaefer M., Valverde MA. BioCreative - Critical Assessment of Information Extraction systems in Biology miguel.valverde@upf.edu 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 fret fluorescent resonance energy transfer FRET RET FRET analysis FRET RET FRET analysis fret predetermined predetermined participant predetermined Arniges M., Fernandez-Fernandez JM., Albrecht N., Schaefer M., Valverde MA. BioCreative - Critical Assessment of Information Extraction systems in Biology miguel.valverde@upf.edu Accepted 2006-AUG-4 by JYOTI. 24-JAN-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation Human TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and trafficking. Human TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and trafficking. J. Biol. Chem. (0021-9258) 2006 imex curation Arniges M., Fernandez-Fernandez JM., Albrecht N., Schaefer M., Valverde MA. BioCreative - Critical Assessment of Information Extraction systems in Biology miguel.valverde@upf.edu 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Arniges M., Fernandez-Fernandez JM., Albrecht N., Schaefer M., Valverde MA. BioCreative - Critical Assessment of Information Extraction systems in Biology miguel.valverde@upf.edu Accepted 2006-AUG-4 by JYOTI. 24-JAN-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation Human TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and trafficking. Human TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and trafficking. J. Biol. Chem. (0021-9258) 2006 imex curation Arniges M., Fernandez-Fernandez JM., Albrecht N., Schaefer M., Valverde MA. BioCreative - Critical Assessment of Information Extraction systems in Biology miguel.valverde@upf.edu 24-JAN-2007: Contacted by JYOTI. Only protein-protein interactions human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. confocal microscopy confocal microscopy antibody detection identification by antibody antibody detection Arniges M., Fernandez-Fernandez JM., Albrecht N., Schaefer M., Valverde MA. BioCreative - Critical Assessment of Information Extraction systems in Biology miguel.valverde@upf.edu Accepted 2006-AUG-4 by JYOTI. 24-JAN-2007: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation trpv4_human Transient receptor potential cation channel subfamily V member 4 TRPV4 Osm-9-like TRP channel 4 Vanilloid receptor-like channel 2 Vanilloid receptor-like protein 2 Vanilloid receptor-related osmotically-activated channel Transient receptor potential protein 12 VRL2 VROAC protein protein human Homo sapiens Human MADSSEGPRAGPGEVAELPGDESGTPGGEAFPLSSLANLFEGEDGSLSPSPADASRPAGPGDGRPNLRMKFQGAFRKGVPNPIDLLESTLYESSVVPGPKKAPMDSLFDYGTYRHHSSDNKRWRKKIIEKQPQSPKAPAPQPPPILKVFNRPILFDIVSRGSTADLDGLLPFLLTHKKRLTDEEFREPSTGKTCLPKALLNLSNGRNDTIPVLLDIAERTGNMREFINSPFRDIYYRGQTALHIAIERRCKHYVELLVAQGADVHAQARGRFFQPKDEGGYFYFGELPLSLAACTNQPHIVNYLTENPHKKADMRRQDSRGNTVLHALVAIADNTRENTKFVTKMYDLLLLKCARLFPDSNLEAVLNNDGLSPLMMAAKTGKIGIFQHIIRREVTDEDTRHLSRKFKDWAYGPVYSSLYDLSSLDTCGEEASVLEILVYNSKIENRHEMLAVEPINELLRDKWRKFGAVSFYINVVSYLCAMVIFTLTAYYQPLEGTPPYPYRTTVDYLRLAGEVITLFTGVLFFFTNIKDLFMKKCPGVNSLFIDGSFQLLYFIYSVLVIVSAALYLAGIEAYLAVMVFALVLGWMNALYFTRGLKLTGTYSIMIQKILFKDLFRFLLVYLLFMIGYASALVSLLNPCANMKVCNEDQTNCTVPTYPSCRDSETFSTFLLDLFKLTIGMGDLEMLSSTKYPVVFIILLVTYIILTFVLLLNMLIALMGETVGQVSKESKHIWKLQWATTILDIERSFPVFLRKAFRSGEMVTVGKSSDGTPDRRWCFRVDEVNWSHWNQNLGIINEDPGKNETYQYYGFSHTVGRLRRDRWSSVVPRVVELNKNSNPDEVVVPLDSMGNPRCDGHQQGYPRKWRTDDAPL DIP protein Q9HBA0 original sequence version: 98 DIP protein Q9HBA0 original sequence version: 131 C62056B86C5A6FB6 q9hba0-1 Transient receptor potential cation channel subfamily V member 4 A Vanilloid receptor-like protein 2 Vanilloid receptor-related osmotically-activated channel TRPV4 VRL2 VROAC Osm-9-like TRP channel 4 Transient receptor potential protein 12 Vanilloid receptor-like channel 2 protein protein human Homo sapiens Human MADSSEGPRAGPGEVAELPGDESGTPGGEAFPLSSLANLFEGEDGSLSPSPADASRPAGPGDGRPNLRMKFQGAFRKGVPNPIDLLESTLYESSVVPGPKKAPMDSLFDYGTYRHHSSDNKRWRKKIIEKQPQSPKAPAPQPPPILKVFNRPILFDIVSRGSTADLDGLLPFLLTHKKRLTDEEFREPSTGKTCLPKALLNLSNGRNDTIPVLLDIAERTGNMREFINSPFRDIYYRGQTALHIAIERRCKHYVELLVAQGADVHAQARGRFFQPKDEGGYFYFGELPLSLAACTNQPHIVNYLTENPHKKADMRRQDSRGNTVLHALVAIADNTRENTKFVTKMYDLLLLKCARLFPDSNLEAVLNNDGLSPLMMAAKTGKIGIFQHIIRREVTDEDTRHLSRKFKDWAYGPVYSSLYDLSSLDTCGEEASVLEILVYNSKIENRHEMLAVEPINELLRDKWRKFGAVSFYINVVSYLCAMVIFTLTAYYQPLEGTPPYPYRTTVDYLRLAGEVITLFTGVLFFFTNIKDLFMKKCPGVNSLFIDGSFQLLYFIYSVLVIVSAALYLAGIEAYLAVMVFALVLGWMNALYFTRGLKLTGTYSIMIQKILFKDLFRFLLVYLLFMIGYASALVSLLNPCANMKVCNEDQTNCTVPTYPSCRDSETFSTFLLDLFKLTIGMGDLEMLSSTKYPVVFIILLVTYIILTFVLLLNMLIALMGETVGQVSKESKHIWKLQWATTILDIERSFPVFLRKAFRSGEMVTVGKSSDGTPDRRWCFRVDEVNWSHWNQNLGIINEDPGKNETYQYYGFSHTVGRLRRDRWSSVVPRVVELNKNSNPDEVVVPLDSMGNPRCDGHQQGYPRKWRTDDAPL C62056B86C5A6FB6 calx_human Calnexin CANX Major histocompatibility complex class I antigen-binding protein p88 p90 IP90 protein protein human Homo sapiens Human MEGKWLLCMLLVLGTAIVEAHDGHDDDVIDIEDDLDDVIEEVEDSKPDTTAPPSSPKVTYKAPVPTGEVYFADSFDRGTLSGWILSKAKKDDTDDEIAKYDGKWEVEEMKESKLPGDKGLVLMSRAKHHAISAKLNKPFLFDTKPLIVQYEVNFQNGIECGGAYVKLLSKTPELNLDQFHDKTPYTIMFGPDKCGEDYKLHFIFRHKNPKTGIYEEKHAKRPDADLKTYFTDKKTHLYTLILNPDNSFEILVDQSVVNSGNLLNDMTPPVNPSREIEDPEDRKPEDWDERPKIPDPEAVKPDDWDEDAPAKIPDEEATKPEGWLDDEPEYVPDPDAEKPEDWDEDMDGEWEAPQIANPRCESAPGCGVWQRPVIDNPNYKGKWKPPMIDNPSYQGIWKPRKIPNPDFFEDLEPFRMTPFSAIGLELWSMTSDIFFDNFIICADRRIVDDWANDGWGLKKAADGAAEPGVVGQMIEAAEERPWLWVVYILTVALPVFLVILFCCSGKKQTSGMEYKKTDAPQPDVKEEEEEKEEEKDKGDEEEEGEEKLEEKQKSDAEEDGGTVSQEEEDRKPKAEEDEILNRSPRNRKPRRE DIP protein P27824 original sequence version: 177 EDE094D9B82261EE q9hba0-2 Transient receptor potential cation channel subfamily V member 4 OTRPC4beta B TRPV4 VRL2 VROAC Osm-9-like TRP channel 4 Transient receptor potential protein 12 Vanilloid receptor-like channel 2 Vanilloid receptor-like protein 2 Vanilloid receptor-related osmotically-activated channel protein protein human Homo sapiens Human MADSSEGPRAGPGEVAELPGDESGTPGGEAFPLSSLANLFEGEDGSLSPSPADASRPAGPGDGRPNLRMKFQGAFRKGVPNPIDLLESTLYESSVVPGPKKAPMDSLFDYGTYRHHSSDNKRWRKKIIEKQPQSPKAPAPQPPPILKVFNRPILFDIVSRGSTADLDGLLPFLLTHKKRLTDEEFREPSTGKTCLPKALLNLSNGRNDTIPVLLDIAERTGNMREFINSPFRDIYYRGQTALHIAIERRCKHYVELLVAQGADVHAQARGRFFQPKDEGGYFYFGELPLSLAACTNQPHIVNYLTENPHKKADMRRQDSRGNTVLHALVAIADNTRENTKFVTKMYDLLLLKCARLFPDSNLEAVLNNDGLSPLMMAAKTGKIGNRHEMLAVEPINELLRDKWRKFGAVSFYINVVSYLCAMVIFTLTAYYQPLEGTPPYPYRTTVDYLRLAGEVITLFTGVLFFFTNIKDLFMKKCPGVNSLFIDGSFQLLYFIYSVLVIVSAALYLAGIEAYLAVMVFALVLGWMNALYFTRGLKLTGTYSIMIQKILFKDLFRFLLVYLLFMIGYASALVSLLNPCANMKVCNEDQTNCTVPTYPSCRDSETFSTFLLDLFKLTIGMGDLEMLSSTKYPVVFIILLVTYIILTFVLLLNMLIALMGETVGQVSKESKHIWKLQWATTILDIERSFPVFLRKAFRSGEMVTVGKSSDGTPDRRWCFRVDEVNWSHWNQNLGIINEDPGKNETYQYYGFSHTVGRLRRDRWSSVVPRVVELNKNSNPDEVVVPLDSMGNPRCDGHQQGYPRKWRTDDAPL Lacks channel activity, due to impaired oligomerization and intracellular retention. A498FF4BBA1CD7A5 q9hba0-5 Transient receptor potential cation channel subfamily V member 4 D TRPV4 VRL2 VROAC Osm-9-like TRP channel 4 Transient receptor potential protein 12 Vanilloid receptor-like channel 2 Vanilloid receptor-like protein 2 Vanilloid receptor-related osmotically-activated channel protein protein human Homo sapiens Human MADSSEGPRAGPGEVAELPGDESGTPGDGRPNLRMKFQGAFRKGVPNPIDLLESTLYESSVVPGPKKAPMDSLFDYGTYRHHSSDNKRWRKKIIEKQPQSPKAPAPQPPPILKVFNRPILFDIVSRGSTADLDGLLPFLLTHKKRLTDEEFREPSTGKTCLPKALLNLSNGRNDTIPVLLDIAERTGNMREFINSPFRDIYYRGQTALHIAIERRCKHYVELLVAQGADVHAQARGRFFQPKDEGGYFYFGELPLSLAACTNQPHIVNYLTENPHKKADMRRQDSRGNTVLHALVAIADNTRENTKFVTKMYDLLLLKCARLFPDSNLEAVLNNDGLSPLMMAAKTGKIGIFQHIIRREVTDEDTRHLSRKFKDWAYGPVYSSLYDLSSLDTCGEEASVLEILVYNSKIENRHEMLAVEPINELLRDKWRKFGAVSFYINVVSYLCAMVIFTLTAYYQPLEGTPPYPYRTTVDYLRLAGEVITLFTGVLFFFTNIKDLFMKKCPGVNSLFIDGSFQLLYFIYSVLVIVSAALYLAGIEAYLAVMVFALVLGWMNALYFTRGLKLTGTYSIMIQKILFKDLFRFLLVYLLFMIGYASALVSLLNPCANMKVCNEDQTNCTVPTYPSCRDSETFSTFLLDLFKLTIGMGDLEMLSSTKYPVVFIILLVTYIILTFVLLLNMLIALMGETVGQVSKESKHIWKLQWATTILDIERSFPVFLRKAFRSGEMVTVGKSSDGTPDRRWCFRVDEVNWSHWNQNLGIINEDPGKNETYQYYGFSHTVGRLRRDRWSSVVPRVVELNKNSNPDEVVVPLDSMGNPRCDGHQQGYPRKWRTDDAPL Forms active ion channels. 1538A7B2E76C8CDF q9hba0-4 Transient receptor potential cation channel subfamily V member 4 TRPV4 VRL2 VROAC Osm-9-like TRP channel 4 Transient receptor potential protein 12 Vanilloid receptor-like channel 2 Vanilloid receptor-like protein 2 Vanilloid receptor-related osmotically-activated channel C protein protein human Homo sapiens Human MADSSEGPRAGPGEVAELPGDESGTPGGEAFPLSSLANLFEGEDGSLSPSPADASRPAGPGDGRPNLRMKFQGAFRKGVPNPIDLLESTLYESSVVPGPKKAPMDSLFDYGTYRHHSSDNKRWRKKIIEKQPQSPKAPAPQPPPILKVFNRPILFDIVSRGSTADLDGLLPFLLTHKKRLTDEEFREPSTGKTCLPKALLNLSNGRNDTIPVLLDIAERTGNMREFINSPFRDIYYRGELPLSLAACTNQPHIVNYLTENPHKKADMRRQDSRGNTVLHALVAIADNTRENTKFVTKMYDLLLLKCARLFPDSNLEAVLNNDGLSPLMMAAKTGKIGIFQHIIRREVTDEDTRHLSRKFKDWAYGPVYSSLYDLSSLDTCGEEASVLEILVYNSKIENRHEMLAVEPINELLRDKWRKFGAVSFYINVVSYLCAMVIFTLTAYYQPLEGTPPYPYRTTVDYLRLAGEVITLFTGVLFFFTNIKDLFMKKCPGVNSLFIDGSFQLLYFIYSVLVIVSAALYLAGIEAYLAVMVFALVLGWMNALYFTRGLKLTGTYSIMIQKILFKDLFRFLLVYLLFMIGYASALVSLLNPCANMKVCNEDQTNCTVPTYPSCRDSETFSTFLLDLFKLTIGMGDLEMLSSTKYPVVFIILLVTYIILTFVLLLNMLIALMGETVGQVSKESKHIWKLQWATTILDIERSFPVFLRKAFRSGEMVTVGKSSDGTPDRRWCFRVDEVNWSHWNQNLGIINEDPGKNETYQYYGFSHTVGRLRRDRWSSVVPRVVELNKNSNPDEVVVPLDSMGNPRCDGHQQGYPRKWRTDDAPL Lacks channel activity, due to impaired oligomerization and intracellular retention. EA7AFC7497D14495 trpv4-canx-1 1 7 unspecified role unspecified role neutral component neutral component 8 unspecified role unspecified role neutral component neutral component colocalization colocalization 3C To determine whether B, C, and E variants were trapped in the ER, we analyzed their glycosylation profile and carried out colocalization studies with calnexin (an ER resident protein) in transfected HeLa cells. trpv4-canx-2 1 7 unspecified role unspecified role neutral component neutral component 10 unspecified role unspecified role neutral component neutral component colocalization colocalization Revisit when isoform are merged in SP 3C To determine whether B, C, and E variants were trapped in the ER, we analyzed their glycosylation profile and carried out colocalization studies with calnexin (an ER resident protein) in transfected HeLa cells. trpv4-canx-3 1 5 unspecified role unspecified role neutral component neutral component 7 unspecified role unspecified role neutral component neutral component colocalization colocalization Revisit when isoform are merged in SP To determine whether B, C, and E variants were trapped in the ER, we analyzed their glycosylation profile and carried out colocalization studies with calnexin (an ER resident protein) in transfected HeLa cells. 3C Splice isoform E trpv4-trpv4-1 2 6 unspecified role unspecified role fluorescence donor fluorescence donor cfp tagged region tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 6 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept region yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined proximity proximity close-contact colocalization neighbourhood interaction 5 FRET results indicate that only TRPV4-A and TRPV4-D efficiently assemble into homomeric channel complexes. To check whether TRPV4 channels oligomerize in the ER we trpv4-trpv4-2 2 9 unspecified role unspecified role fluorescence donor fluorescence donor cfp tagged region tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 9 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept region yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined proximity proximity close-contact colocalization neighbourhood interaction 5 FRET results indicate that only TRPV4-A and TRPV4-D efficiently assemble into homomeric channel complexes. To check whether TRPV4 channels oligomerize in the ER we trpv4-trpv4-3 4 9 unspecified role unspecified role neutral component neutral component 6 unspecified role unspecified role neutral component neutral component colocalization colocalization 6A As shown in Fig. 6Aplasmamembrane overlapping was only observed in cells expressing TRPV4-A and TRPV4-D (with almost identical localization), Splice isoform D colocalises withQ9HBA0 trpv4-trpv4-4 2 6 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence accept region yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 9 unspecified role unspecified role fluorescence donor fluorescence donor cfp tagged region tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined proximity proximity close-contact colocalization neighbourhood interaction 6B Using TRPV4-A-YFP as the FRETacceptor and CFP-fused TRPV4-B-E variants as donors, a high FRET efficency of 21.7 3.5 could only be demonstrated for the heteromeric channel formation between TRPV4-A and TRPV4-D subunits. trpv4-trpv4-5 3 9 unspecified role unspecified role prey prey c-terminal yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 6 unspecified role unspecified role bait bait c-terminal flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range physical association physical association 6C From all the combinations tested with the TRPV4-A-FLAG-tagged protein coexpressed with TRPV4-YFP isoforms in HEK-293 cells, we were able to coimmunoprecipitate only the TRPV4-D variant (Fig. 6C, top panel).