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/ http://www.ebi.ac.uk/ ban-2018-1 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 The Biochemical journal 2018 Only protein-protein interactions imex curation imex curation ban-2018-2 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 The Biochemical journal 2018 Only protein-protein interactions imex curation imex curation ban-2018-3 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 The Biochemical journal 2018 Only protein-protein interactions imex curation imex curation 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 arrb2_human Beta-arrestin-2 Arrestin beta-2 ARRB2 ARB2 ARR2 Non-visual arrestin-3 protein protein human Homo sapiens Human MGEKPGTRVFKKSSPNCKLTVYLGKRDFVDHLDKVDPVDGVVLVDPDYLKDRKVFVTLTCAFRYGREDLDVLGLSFRKDLFIATYQAFPPVPNPPRPPTRLQDRLLRKLGQHAHPFFFTIPQNLPCSVTLQPGPEDTGKACGVDFEIRAFCAKSLEEKSHKRNSVRLVIRKVQFAPEKPGPQPSAETTRHFLMSDRSLHLEASLDKELYYHGEPLNVNVHVTNNSTKTVKKIKVSVRQYADICLFSTAQYKCPVAQLEQDDQVSPSSTFCKVYTITPLLSDNREKRGLALDGKLKHEDTNLASSTIVKEGANKEVLGILVSYRVKVKLVVSRGGDVSVELPFVLMHPKPHDHIPLPRPQSAAPETDVPVDTNLIEFDTNYATDDDIVFEDFARLRLKGMKDDDYDDQLC arbk1_human Beta-adrenergic receptor kinase 1 GRK2 ADRBK1 BARK BARK1 G-protein coupled receptor kinase 2 protein protein human Homo sapiens Human MADLEAVLADVSYLMAMEKSKATPAARASKKILLPEPSIRSVMQKYLEDRGEVTFEKIFSQKLGYLLFRDFCLNHLEEARPLVEFYEEIKKYEKLETEEERVARSREIFDSYIMKELLACSHPFSKSATEHVQGHLGKKQVPPDLFQPYIEEICQNLRGDVFQKFIESDKFTRFCQWKNVELNIHLTMNDFSVHRIIGRGGFGEVYGCRKADTGKMYAMKCLDKKRIKMKQGETLALNERIMLSLVSTGDCPFIVCMSYAFHTPDKLSFILDLMNGGDLHYHLSQHGVFSEADMRFYAAEIILGLEHMHNRFVVYRDLKPANILLDEHGHVRISDLGLACDFSKKKPHASVGTHGYMAPEVLQKGVAYDSSADWFSLGCMLFKLLRGHSPFRQHKTKDKHEIDRMTLTMAVELPDSFSPELRSLLEGLLQRDVNRRLGCLGRGAQEVKESPFFRSLDWQMVFLQKYPPPLIPPRGEVNAADAFDIGSFDEEDTKGIKLLDSDQELYRNFPLTISERWQQEVAETVFDTINAETDRLEARKKAKNKQLGHEEDYALGKDCIMHGYMSKMGNPFLTQWQRRYFYLFPNRLEWRGEGEAPQSLLTMEEIQSVEETQIKERKCLLLKIRGGKQFILQCDSDPELVQWKKELRDAYREAQQLVQRVPKMKNKPRSPVVELSKVPLVQRGSANGL aplnr-arrb2-1 3555919 N/A 3555922 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 false N/A 3555923 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 false physical association physical association false false false Figure 3A 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 3555920 N/A 3555922 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 false P35414:p.Ile109Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false P35414:p.Arg168Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false N/A 3555924 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 false proximity proximity close-contact colocalization neighbourhood interaction false false false Figure 3B Apelin-13 peptide aplnr-arrb2-2 3555921 N/A 3555922 immunocytochemistry immunocytochemistry 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 false 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 false N/A 3555923 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 false colocalization colocalization false false false 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