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/ Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle. Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle. Open biology 2018 imex curation Albee LJ., LaPorte HM., Gao X., Eby JM., Cheng YH., Nevins AM., Volkman BF., Gaponenko V., Majetschak M. mmajetschak@luc.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. pla proximity ligation assay pELISA pELISA PLA antibody detection identification by antibody antibody detection Albee LJ., LaPorte HM., Gao X., Eby JM., Cheng YH., Nevins AM., Volkman BF., Gaponenko V., Majetschak M. mmajetschak@luc.edu Accepted 2019-JUN-13 AT 10:12 BST AT 10:12 BST by PPORRAS Open biology 2018 Only protein-protein interactions imex curation imex curation I agree. I have deleted the interaction (figure 3C) and added the figure legend to EBI-21299013 in Expt-4. Please delete this experiment after reading this! Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle. Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle. Open biology 2018 imex curation Albee LJ., LaPorte HM., Gao X., Eby JM., Cheng YH., Nevins AM., Volkman BF., Gaponenko V., Majetschak M. mmajetschak@luc.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 anti tag western anti tag western blot anti tag western Albee LJ., LaPorte HM., Gao X., Eby JM., Cheng YH., Nevins AM., Volkman BF., Gaponenko V., Majetschak M. mmajetschak@luc.edu Accepted 2019-JUN-13 AT 10:12 BST AT 10:12 BST by PPORRAS Open biology 2018 Only protein-protein interactions imex curation imex curation I agree. I have deleted the interaction (figure 3C) and added the figure legend to EBI-21299013 in Expt-4. Please delete this experiment after reading this! Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle. Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle. Open biology 2018 imex curation Albee LJ., LaPorte HM., Gao X., Eby JM., Cheng YH., Nevins AM., Volkman BF., Gaponenko V., Majetschak M. mmajetschak@luc.edu 25-Jul-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-smooth muscle Homo sapiens smooth_muscle smooth muscle pla proximity ligation assay pELISA pELISA PLA antibody detection identification by antibody antibody detection Albee LJ., LaPorte HM., Gao X., Eby JM., Cheng YH., Nevins AM., Volkman BF., Gaponenko V., Majetschak M. primary aortic smooth muscle cells mmajetschak@luc.edu Accepted 2019-JUN-13 AT 10:12 BST AT 10:12 BST by PPORRAS Open biology 2018 Only protein-protein interactions imex curation imex curation I agree. I have deleted the interaction (figure 3C) and added the figure legend to EBI-21299013 in Expt-4. Please delete this experiment after reading this! Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle. Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle. Open biology 2018 imex curation Albee LJ., LaPorte HM., Gao X., Eby JM., Cheng YH., Nevins AM., Volkman BF., Gaponenko V., Majetschak M. mmajetschak@luc.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 Albee LJ., LaPorte HM., Gao X., Eby JM., Cheng YH., Nevins AM., Volkman BF., Gaponenko V., Majetschak M. mmajetschak@luc.edu Accepted 2019-JUN-13 AT 10:12 BST AT 10:12 BST by PPORRAS Open biology 2018 Only protein-protein interactions imex curation imex curation I agree. I have deleted the interaction (figure 3C) and added the figure legend to EBI-21299013 in Expt-4. Please delete this experiment after reading this! Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle. Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle. Open biology 2018 imex curation Albee LJ., LaPorte HM., Gao X., Eby JM., Cheng YH., Nevins AM., Volkman BF., Gaponenko V., Majetschak M. mmajetschak@luc.edu 25-Jul-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation rat-a7r5 Rattus norvegicus a7r5 a7r5 pla proximity ligation assay pELISA pELISA PLA antibody detection identification by antibody antibody detection Albee LJ., LaPorte HM., Gao X., Eby JM., Cheng YH., Nevins AM., Volkman BF., Gaponenko V., Majetschak M. mmajetschak@luc.edu Accepted 2019-JUN-13 AT 10:12 BST AT 10:12 BST by PPORRAS Open biology 2018 Only protein-protein interactions imex curation imex curation I agree. I have deleted the interaction (figure 3C) and added the figure legend to EBI-21299013 in Expt-4. Please delete this experiment after reading this! Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle. Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle. Open biology 2018 imex curation Albee LJ., LaPorte HM., Gao X., Eby JM., Cheng YH., Nevins AM., Volkman BF., Gaponenko V., Majetschak M. mmajetschak@luc.edu 25-Jul-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-smooth muscle Homo sapiens smooth_muscle smooth muscle anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Albee LJ., LaPorte HM., Gao X., Eby JM., Cheng YH., Nevins AM., Volkman BF., Gaponenko V., Majetschak M. primary aortic smooth muscle cells mmajetschak@luc.edu Accepted 2019-JUN-13 AT 10:12 BST AT 10:12 BST by PPORRAS Open biology 2018 Only protein-protein interactions imex curation imex curation I agree. I have deleted the interaction (figure 3C) and added the figure legend to EBI-21299013 in Expt-4. Please delete this experiment after reading this! ackr3_human Atypical chemokine receptor 3 ACKR3 C-X-C chemokine receptor type 7 CMKOR1 CXCR7 Chemokine orphan receptor 1 G-protein coupled receptor 159 G-protein coupled receptor RDC1 homolog GPR159 RDC1 protein protein human Homo sapiens Human MDLHLFDYSEPGNFSDISWPCNSSDCIVVDTVMCPNMPNKSVLLYTLSFIYIFIFVIGMIANSVVVWVNIQAKTTGYDTHCYILNLAIADLWVVLTIPVWVVSLVQHNQWPMGELTCKVTHLIFSINLFGSIFFLTCMSVDRYLSITYFTNTPSSRKKMVRRVVCILVWLLAFCVSLPDTYYLKTVTSASNNETYCRSFYPEHSIKEWLIGMELVSVVLGFAVPFSIIAVFYFLLARAISASSDQEKHSSRKIIFSYVVVFLVCWLPYHVAVLLDIFSILHYIPFTCRLEHALFTALHVTQCLSLVHCCVNPVLYSFINRNYRYELMKAFIFKYSAKTGLTKLIDASRVSETEYSALEQSTK A863EC1AFB5B158B tm7_peptide TM7_peptide peptide peptide polypeptide oligopeptide oligopeptide polypeptide chemical synthesis Chemical synthesis (Chemical synthesis) DDDLHVTQCLSLVHCCVNPVLYSFIN cxcr4_human C-X-C chemokine receptor type 4 Stromal cell-derived factor 1 receptor Fusin Leukocyte-derived seven transmembrane domain receptor LCR1 FB22 NPYRL HM89 CXCR4 Lipopolysaccharide-associated protein 3 protein protein human Homo sapiens Human MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS DIP protein P61073 original sequence version: 80 8C8476A186786B83 v1ar_human Vasopressin V1a receptor AVPR1A AVPR1 AVPR V1a Antidiuretic hormone receptor 1a Vascular/hepatic-type arginine vasopressin receptor protein protein human Homo sapiens Human MRLSAGPDAGPSGNSSPWWPLATGAGNTSREAEALGEGNGPPRDVRNEELAKLEIAVLAVTFAVAVLGNSSVLLALHRTPRKTSRMHLFIRHLSLADLAVAFFQVLPQMCWDITYRFRGPDWLCRVVKHLQVFGMFASAYMLVVMTADRYIAVCHPLKTLQQPARRSRLMIAAAWVLSFVLSTPQYFVFSMIEVNNVTKARDCWATFIQPWGSRAYVTWMTGGIFVAPVVILGTCYGFICYNIWCNVRGKTASRQSKGAEQAGVAFQKGFLLAPCVSSVKSISRAKIRTVKMTFVIVTAYIVCWAPFFIIQMWSVWDPMSVWTESENPTITITALLGSLNSCCNPWIYMFFSGHLLQDCVQSFPCCQNMKEKFNKEDTDSMSRRQTFYSNNRSPTNSTGMWKDSPKSSKSIKFIPVST 477D4260EC826561 ackr3_rat Atypical chemokine receptor 3 Ackr3 Cmkor1 Cxcr7 Rdc1 C-X-C chemokine receptor type 7 Chemokine orphan receptor 1 G-protein coupled receptor RDC1 homolog protein protein rat Rattus norvegicus (Rat) Rat MDVHLFDYVEPGNYSDINWPCNSSDCIVVDTVQCPAMPNKNVLLYTLSFIYIFIFVIGMIANSVVVWVNIQAKTTGYDTHCYILNLAIADLWVVITIPVWVVSLVQHNQWPMGELTCKITHLIFSINLFGSIFFLACMSVDRYLSITYFTSTSSYKKKMVRRVVCVLVWLLAFFVSLPDTYYLKTVTSASNNETYCRSFYPEHSIKEWLIGMELVSVILGFAVPFTIIAIFYFLLARAMSASGDQEKHSSRKIIFSYVVVFLVCWLPYHFVVLLDIFSILHYIPFTCQLENVLFTALHVTQCLSLVHCCVNPVLYSFINRNYRYELMKAFIFKYSAKTGLTKLIDASRVSETEYSALEQNTK C47D1DD678697CFD cxcr4_rat C-X-C chemokine receptor type 4 Cxcr4 Cmkar4 Fusin Leukocyte-derived seven transmembrane domain receptor Stromal cell-derived factor 1 receptor protein protein rat Rattus norvegicus (Rat) Rat MEIYTSDNYSEEVGSGDYDSNKEPCFRDENENFNRIFLPTIYFIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAMADWYFGKFLCKAVHIIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKAVYVGVWIPALLLTIPDIIFADVSQGDGRYICDRLYPDSLWMVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYVGISIDSFILLEVIKQGCEFESVVHKWISITEALAFFHCCLNPILYAFLGAKFKSSAQHALNSMSRGSSLKILSKGKRGGHSSVSTESESSSFHSS 09D19860D3D2CB8A tm4_peptide TM4_peptide peptide peptide polypeptide oligopeptide oligopeptide polypeptide chemical synthesis Chemical synthesis (Chemical synthesis) VVCILWLLAFCVSLPDTYYLDD v1ar_rat Vasopressin V1a receptor Avpr1a AVPR V1a Antidiuretic hormone receptor 1a Vascular/hepatic-type arginine vasopressin receptor protein protein rat Rattus norvegicus (Rat) Rat MSFPRGSQDRSVGNSSPWWPLTTEGSNGSQEAARLGEGDSPLGDVRNEELAKLEIAVLAVIFVVAVLGNSSVLLALHRTPRKTSRMHLFIRHLSLADLAVAFFQVLPQLCWDITYRFRGPDWLCRVVKHLQVFAMFASAYMLVVMTADRYIAVCHPLKTLQQPARRSRLMIATSWVLSFILSTPQYFIFSVIEIEVNNGTKTQDCWATFIQPWGTRAYVTWMTSGVFVAPVVVLGTCYGFICYHIWRNIRGKTASSRHSKGDKGSGEAVGPFHKGLLVTPCVSSVKSISRAKIRTVKMTFVIVSAYILCWAPFFIVQMWSVWDENFIWTDSENPSITITALLASLNSCCNPWIYMFFSGHLLQDCVQSFPCCHSMAQKFAKDDSDSMSRRQTSYSNNRSPTNSTGMWKDSPKSSKSIRFIPVST 4F452C8DF7B4C900 tm2_peptide TM2_peptide peptide peptide polypeptide oligopeptide oligopeptide polypeptide chemical synthesis Chemical synthesis (Chemical synthesis) YILNLAIADLWVVLTIPVWVVDDD ackr3-avpr1a-1 1 10 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed 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 n-terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range proximity proximity close-contact colocalization neighbourhood interaction Figure 2A/B ackr3-avpr1a-2 2 10 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed 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 prey prey over-expressed over expressed level over-expressed n-terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association Figure 2C avpr1a-ackr3-1 4 10 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 eyfp enhanced yellow fluorescent protein tag eyfp 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 2D/E ackr3-avpr1a-3 3 7 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level 10 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level proximity proximity close-contact colocalization neighbourhood interaction Figure 3 A/B/C, 4 B/D, 6A/B, 7, 8 A/B Signals corresponding to ACKR3 : AVPR1A and ACKR3 : CXCR4 heteromers in hVSMC incubated with ACKR3 siRNA were reduced when compared with hVSMC incubated with NT-siRNA. CXCR4 silencing did not affect the number of PLA signals for ACKR3 : AVPR1A interactions. ackr3-avpr1a-5 6 9 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level 7 unspecified role unspecified role prey prey physiological level physiological level endogenous level endogenous endogenous endogenous level 10 unspecified role unspecified role bait bait physiological level physiological level endogenous level endogenous endogenous endogenous level association association Figure 3D-G ackr3-avpr1a-6 3 9 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level 10 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level proximity proximity close-contact colocalization neighbourhood interaction Figure 3A/B,4B/D, 6A/B, 7, 8A/B ACKR3 -Uniprot P25106 As shown In figure 3, signals for CXCR4 : AVPR1A interactions were indistinguishable from negative control experiments. When hVSMCs were incubated with ACKR3 siRNA, PLA signals corresponding to CXCR4 : AVPR1A interactions increased to 510% of PLA signals in hVSMC incubated with NT-siRNA. Addition of TM 2/4/7 (ACKR3) peptides had no effect on this interaction. ackr3-avpr1a-7 3 7 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level 9 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level proximity proximity close-contact colocalization neighbourhood interaction Figure 4 B/D, 7,8 A/B Signals corresponding to ACKR3 : AVPR1A and ACKR3 : CXCR4 heteromers in hVSMC incubated with ACKR3 siRNA were reduced when compared with hVSMC incubated with NT-siRNA. Weak interaction ackr3-avpr1a-8 5 11 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level 14 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level proximity proximity close-contact colocalization neighbourhood interaction Figure 5 B/D Signals corresponding to ACKR3 : AVPR1A and ACKR3 : CXCR4 heteromers in cells incubated with ACKR3 siRNA were reduced when compared with cells incubated with NT-siRNA. ackr3-avpr1a-9 5 11 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level 12 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level proximity proximity close-contact colocalization neighbourhood interaction Figure 5 B/D Signals corresponding to ACKR3 : AVPR1A and ACKR3 : CXCR4 heteromers in cells incubated with ACKR3 siRNA were reduced when compared with cells incubated with NT-siRNA. avpr1a-cxcr4-1 5 12 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level 14 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level proximity proximity close-contact colocalization neighbourhood interaction Figure 5 B/D ACKR3 -Uniprot O89039 When cells were incubated with ACKR3 siRNA, PLA signals corresponding to CXCR4 : AVPR1A interactions increased to 276% of PLA signals in cells incubated with NT-siRNA. ackr3-avpr1a-10 3 15 competitor competitor competitor neutral component neutral component purified purified chemical synthesis Chemical synthesis (Chemical synthesis) 7 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level 10 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level proximity proximity close-contact colocalization neighbourhood interaction Figure 7,8 A/B The TM2 and TM4 peptides reduced PLA signals for AVPR1A : ACKR3 interactions. ackr3-avpr1a-11 3 13 competitor competitor competitor neutral component neutral component purified purified chemical synthesis Chemical synthesis (Chemical synthesis) 7 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level 10 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level proximity proximity close-contact colocalization neighbourhood interaction Figure 7,8 A/Bi The TM2 and TM4 peptides reduced PLA signals for AVPR1A : ACKR3 interactions. ackr3-cxcr4-13 3 15 competitor competitor competitor neutral component neutral component purified purified chemical synthesis Chemical synthesis (Chemical synthesis) 7 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level 9 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level proximity proximity close-contact colocalization neighbourhood interaction Figure 7,8 A/B All TM peptide analogues reduced PLA signals for CXCR4 : ACKR3 interactions. ackr3-cxcr4-15 3 13 competitor competitor competitor neutral component neutral component purified purified chemical synthesis Chemical synthesis (Chemical synthesis) 7 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level 9 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level proximity proximity close-contact colocalization neighbourhood interaction Figure 7,8 A/Bi All TM peptide analogues reduced PLA signals for CXCR4 : ACKR3 interactions. ackr3-cxcr4-16 3 8 competitor competitor competitor neutral component neutral component purified purified chemical synthesis Chemical synthesis (Chemical synthesis) 7 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level 9 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level proximity proximity close-contact colocalization neighbourhood interaction Figure 7,8 A/Bii All TM peptide analogues reduced PLA signals for CXCR4 : ACKR3 interactions.