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/ GPCR structure and function relationship: identification of a biased apelin receptor mutant. GPCR structure and function relationship: identification of a biased apelin receptor mutant. The Biochemical journal 2018 imex curation Ban T., Li X., Ma X., Yang H., Song Y., Sun Y., Shen M., Li N., Zhang MY., Ma Y., Zhong W., Zhang M., Hu LA. Apelin Receptor is of Human origin based on Ref. 21. Beta Arrestin-2 is also of Human origin based on Ref. 24. Apelin-13 peptide has been entered as being of Human origin based on the fact Human, Rat and Mouse sequences are identical. liaoyuan@amgen.com 25-Jul-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-u2-os Human U2-OS osteosarcoma cell line u2-os U2-OS osteocaroma cell line Not in CAPRI and no primary literature reference confocal microscopy confocal microscopy tag fluorescence tag visualisation by fluorescence tag fluorescence Ban T., Li X., Ma X., Yang H., Song Y., Sun Y., Shen M., Li N., Zhang MY., Ma Y., Zhong W., Zhang M., Hu LA. Apelin Receptor is of Human origin based on Ref. 21. Beta Arrestin-2 is also of Human origin based on Ref. 24. Apelin-13 peptide has been entered as being of Human origin based on the fact Human, Rat and Mouse sequences are identical. liaoyuan@amgen.com Accepted 2019-MAY-24 AT 08:29 BST AT 08:29 BST by ORCHARD The Biochemical journal 2018 Only protein-protein interactions imex curation imex curation GPCR structure and function relationship: identification of a biased apelin receptor mutant. GPCR structure and function relationship: identification of a biased apelin receptor mutant. The Biochemical journal 2018 imex curation Ban T., Li X., Ma X., Yang H., Song Y., Sun Y., Shen M., Li N., Zhang MY., Ma Y., Zhong W., Zhang M., Hu LA. Apelin Receptor is of Human origin based on Ref. 21. Beta Arrestin-2 is also of Human origin based on Ref. 24. Apelin-13 peptide has been entered as being of Human origin based on the fact Human, Rat and Mouse sequences are identical. liaoyuan@amgen.com 25-Jul-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-293ft Homo sapiens HEK293FT cell line 293ft HEK293FT cell line bret bioluminescence resonance energy transfer BRET LRET LRET BRET bret tag visualisation tag visualisation Ban T., Li X., Ma X., Yang H., Song Y., Sun Y., Shen M., Li N., Zhang MY., Ma Y., Zhong W., Zhang M., Hu LA. Apelin Receptor is of Human origin based on Ref. 21. Beta Arrestin-2 is also of Human origin based on Ref. 24. Apelin-13 peptide has been entered as being of Human origin based on the fact Human, Rat and Mouse sequences are identical. liaoyuan@amgen.com Accepted 2019-MAY-24 AT 08:29 BST AT 08:29 BST by ORCHARD The Biochemical journal 2018 Only protein-protein interactions imex curation imex curation GPCR structure and function relationship: identification of a biased apelin receptor mutant. GPCR structure and function relationship: identification of a biased apelin receptor mutant. The Biochemical journal 2018 imex curation Ban T., Li X., Ma X., Yang H., Song Y., Sun Y., Shen M., Li N., Zhang MY., Ma Y., Zhong W., Zhang M., Hu LA. Apelin Receptor is of Human origin based on Ref. 21. Beta Arrestin-2 is also of Human origin based on Ref. 24. Apelin-13 peptide has been entered as being of Human origin based on the fact Human, Rat and Mouse sequences are identical. liaoyuan@amgen.com 25-Jul-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-293ft Homo sapiens HEK293FT cell line 293ft HEK293FT cell line split luciferase complementation split luciferase complementation tag luciferase tag visualisation by luciferase assay tag luciferase Ban T., Li X., Ma X., Yang H., Song Y., Sun Y., Shen M., Li N., Zhang MY., Ma Y., Zhong W., Zhang M., Hu LA. NanoBit assay:-Bioluminescence system using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, producing luminescence (signal half-life >2 h) with a specific activity ~150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases. Apelin Receptor is of Human origin based on Ref. 21. Beta Arrestin-2 is also of Human origin based on Ref. 24. Apelin-13 peptide has been entered as being of Human origin based on the fact Human, Rat and Mouse sequences are identical. liaoyuan@amgen.com Accepted 2019-MAY-24 AT 08:29 BST AT 08:29 BST by ORCHARD The Biochemical journal 2018 Only protein-protein interactions imex curation imex curation arrb2_human Beta-arrestin-2 Arrestin beta-2 ARRB2 ARB2 ARR2 Non-visual arrestin-3 protein protein human Homo sapiens Human MGEKPGTRVFKKSSPNCKLTVYLGKRDFVDHLDKVDPVDGVVLVDPDYLKDRKVFVTLTCAFRYGREDLDVLGLSFRKDLFIATYQAFPPVPNPPRPPTRLQDRLLRKLGQHAHPFFFTIPQNLPCSVTLQPGPEDTGKACGVDFEIRAFCAKSLEEKSHKRNSVRLVIRKVQFAPEKPGPQPSAETTRHFLMSDRSLHLEASLDKELYYHGEPLNVNVHVTNNSTKTVKKIKVSVRQYADICLFSTAQYKCPVAQLEQDDQVSPSSTFCKVYTITPLLSDNREKRGLALDGKLKHEDTNLASSTIVKEGANKEVLGILVSYRVKVKLVVSRGGDVSVELPFVLMHPKPHDHIPLPRPQSAAPETDVPVDTNLIEFDTNYATDDDIVFEDFARLRLKGMKDDDYDDQLC DIP protein P32121 original sequence version: 148 DIP protein P32121 original sequence version: 102 DIP protein P32121-1 original sequence version: 86 DIP protein P32121-1 original sequence version: 100 DIP protein P32121 original sequence version: 153 DEEC507D4A7B84FF arbk1_human Beta-adrenergic receptor kinase 1 GRK2 ADRBK1 BARK BARK1 G-protein coupled receptor kinase 2 protein protein human Homo sapiens Human MADLEAVLADVSYLMAMEKSKATPAARASKKILLPEPSIRSVMQKYLEDRGEVTFEKIFSQKLGYLLFRDFCLNHLEEARPLVEFYEEIKKYEKLETEEERVARSREIFDSYIMKELLACSHPFSKSATEHVQGHLGKKQVPPDLFQPYIEEICQNLRGDVFQKFIESDKFTRFCQWKNVELNIHLTMNDFSVHRIIGRGGFGEVYGCRKADTGKMYAMKCLDKKRIKMKQGETLALNERIMLSLVSTGDCPFIVCMSYAFHTPDKLSFILDLMNGGDLHYHLSQHGVFSEADMRFYAAEIILGLEHMHNRFVVYRDLKPANILLDEHGHVRISDLGLACDFSKKKPHASVGTHGYMAPEVLQKGVAYDSSADWFSLGCMLFKLLRGHSPFRQHKTKDKHEIDRMTLTMAVELPDSFSPELRSLLEGLLQRDVNRRLGCLGRGAQEVKESPFFRSLDWQMVFLQKYPPPLIPPRGEVNAADAFDIGSFDEEDTKGIKLLDSDQELYRNFPLTISERWQQEVAETVFDTINAETDRLEARKKAKNKQLGHEEDYALGKDCIMHGYMSKMGNPFLTQWQRRYFYLFPNRLEWRGEGEAPQSLLTMEEIQSVEETQIKERKCLLLKIRGGKQFILQCDSDPELVQWKKELRDAYREAQQLVQRVPKMKNKPRSPVVELSKVPLVQRGSANGL DIP protein P25098 original sequence version: 156 DIP protein P25098 original sequence version: 124 4DB68F62841AFA7D apj_human Apelin receptor APLNR AGTRL1 APJ Angiotensin receptor-like 1 G-protein coupled receptor APJ G-protein coupled receptor HG11 protein protein human Homo sapiens Human MEEGGDFDNYYGADNQSECEYTDWKSSGALIPAIYMLVFLLGTTGNGLVLWTVFRSSREKRRSADIFIASLAVADLTFVVTLPLWATYTYRDYDWPFGTFFCKLSSYLIFVNMYASVFCLTGLSFDRYLAIVRPVANARLRLRVSGAVATAVLWVLAALLAMPVMVLRTTGDLENTTKVQCYMDYSMVATVSSEWAWEVGLGVSSTTVGFVVPFTIMLTCYFFIAQTIAGHFRKERIEGLRKRRRLLSIIVVLVVTFALCWMPYHLVKTLYMLGSLLHWPCDFDLFLMNIFPYCTCISYVNSCLNPFLYAFFDPRFRQACTSMLCCGQSRCAGTSHSSSGEKSASYSSGHSQGPGPNMGKGGEQMHEKSIPYSQETLVVD 78DB18BEA6D2B2E4 aplnr-arrb2-1 3 6 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed small_bit luciferase tag luciferase tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 4 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed large_bit luciferase tag luciferase tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association Figure 3A text states:- the small part of Nanoluc (Smbit) tag was fused to the C-terminal APJ, and the large part of Nanoluc (Lgbit) was fused to the N-terminal β-arrestin. I think this is meant to mean the tags are fused to C/N terminus of the proteins. Apelin-13 peptide Agonist AP-13 stimulation induced a rapid and robust signal increase. Under the same conditions, the APJ I109A mutant completely abolished AP-13 stimulated beta-arrestin recruitment (Fig. 3A) aplnr-grk2-1 2 6 unspecified role unspecified role fluorescence accept fluorescence acceptor fluorescence 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 P35414:p.Ile109Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain I A Mon Apr 29 11:26:48 BST 2019 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Iso109Ala' Tue Jun 30 18:38:36 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ile109Ala' P35414:p.Arg168Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain R A Tue Jun 30 16:08:56 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg168Ala' 5 unspecified role unspecified role fluorescence donor fluorescence donor over-expressed over expressed level over-expressed region renilla luciferase renilla luciferase protein tag renilla luciferase undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined proximity proximity close-contact colocalization neighbourhood interaction Figure 3B Apelin-13 peptide RLuc tag added to GRK2 as there is no mention of a GFP tag for this protein, only RLuc. aplnr-arrb2-2 1 6 immunocytochemistry immunocytochemistry tag fluorescence tag visualisation by fluorescence tag fluorescence 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 P35414:p.Ile109Ala mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain I A Tue Jun 30 18:32:41 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ile109Ala' 4 unspecified role unspecified role neutral component neutral component 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 colocalization colocalization Figure 3 C/D In wild type APJ expressing cells, treatment with AP-13 caused a quick beta-arrestin translocation from cytoplasm to plasma membrane. The APJ/beta-arrestin complexes disappeared from the plasma membrane and were internalized into the cytosol to form large intracellular vesicles at 60 minutes (Fig. 3C). However, AP-13 stimulated beta-arrestin recruitment and receptor internalization were not detected in APJ I109A mutant expressing cells even after agonist treatment for 60 minutes (Fig. 3D) AP-13 peptide I have added the mutation as "no effect" as there appears to be observable colocalisation with WT AND mutant before agonist addition. -Unlike BRET- Fig 3A where there appears to be very little interaction with mutant (or WT) before AP-13 addition..