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/ Lrp4 is a receptor for Agrin and forms a complex with MuSK. Lrp4 is a receptor for Agrin and forms a complex with MuSK. Cell (0092-8674) 2008 imex curation Kim N., Stiegler AL., Cameron TO., Hallock PT., Gomez AM., Huang JH., Hubbard SR., Dustin ML., Burden SJ. BaF3 cells expressing bait proteins on the surface were perfused through a chamber containing a glass coverslip on which Agrin B8 had been adsorbed. Bright field, fluorescence and interference reflection microscopy were used to determine the number of cells that bound to each substrate. burden@saturn.med.nyu.edu 16-Dec-2008: Contacted by JYOTI. davet Only protein-protein interactions 4 in vitro In vitro pull down pull down tag fluorescence tag visualisation by fluorescence tag fluorescence Kim N., Stiegler AL., Cameron TO., Hallock PT., Gomez AM., Huang JH., Hubbard SR., Dustin ML., Burden SJ. BaF3 cells expressing bait proteins on the surface were perfused through a chamber containing a glass coverslip on which Agrin B8 had been adsorbed. Bright field, fluorescence and interference reflection microscopy were used to determine the number of cells that bound to each substrate. burden@saturn.med.nyu.edu Accepted 2008-Not a month!-8 by DAVET. 16-Dec-2008: Contacted by JYOTI. Cell (0092-8674) 2008 davet Only protein-protein interactions 4 imex curation Lrp4 is a receptor for Agrin and forms a complex with MuSK. Lrp4 is a receptor for Agrin and forms a complex with MuSK. Cell (0092-8674) 2008 imex curation Kim N., Stiegler AL., Cameron TO., Hallock PT., Gomez AM., Huang JH., Hubbard SR., Dustin ML., Burden SJ. BaF3 cells expressing bait proteins on the surface were perfused through a chamber containing a glass coverslip on which Agrin B8 had been adsorbed. Bright field, fluorescence and interference reflection microscopy were used to determine the number of cells that bound to each substrate. burden@saturn.med.nyu.edu 16-Dec-2008: Contacted by JYOTI. davet Only protein-protein interactions 4 in vitro In vitro pull down pull down predetermined predetermined participant predetermined Kim N., Stiegler AL., Cameron TO., Hallock PT., Gomez AM., Huang JH., Hubbard SR., Dustin ML., Burden SJ. BaF3 cells expressing bait proteins on the surface were perfused through a chamber containing a glass coverslip on which Agrin B8 had been adsorbed. Bright field, fluorescence and interference reflection microscopy were used to determine the number of cells that bound to each substrate. burden@saturn.med.nyu.edu Accepted 2008-Not a month!-8 by DAVET. 16-Dec-2008: Contacted by JYOTI. Cell (0092-8674) 2008 davet Only protein-protein interactions 4 imex curation Lrp4 is a receptor for Agrin and forms a complex with MuSK. Lrp4 is a receptor for Agrin and forms a complex with MuSK. Cell (0092-8674) 2008 imex curation Kim N., Stiegler AL., Cameron TO., Hallock PT., Gomez AM., Huang JH., Hubbard SR., Dustin ML., Burden SJ. BaF3 cells expressing bait proteins on the surface were perfused through a chamber containing a glass coverslip on which Agrin B8 had been adsorbed. Bright field, fluorescence and interference reflection microscopy were used to determine the number of cells that bound to each substrate. burden@saturn.med.nyu.edu 16-Dec-2008: Contacted by JYOTI. davet Only protein-protein interactions 4 mouse-ba_f3 Mouse pro B cells IL-3 dependent mouse-baf3 EBI-6922762 ba_f3 Mouse pro B cells IL-3 dependent BA/F3 baf3 anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Kim N., Stiegler AL., Cameron TO., Hallock PT., Gomez AM., Huang JH., Hubbard SR., Dustin ML., Burden SJ. BaF3 cells expressing bait proteins on the surface were perfused through a chamber containing a glass coverslip on which Agrin B8 had been adsorbed. Bright field, fluorescence and interference reflection microscopy were used to determine the number of cells that bound to each substrate. burden@saturn.med.nyu.edu Accepted 2008-Not a month!-8 by DAVET. 16-Dec-2008: Contacted by JYOTI. Cell (0092-8674) 2008 davet Only protein-protein interactions 4 imex curation agrin_rat Agrin Agrn Agrin protein protein rat Rattus norvegicus (Rat) Rat MPPLPLEHRPRQEPGASMLVRYFMIPCNICLILLATSTLGFAVLLFLSNYKPGIHFTPAPPTPPDVCRGMLCGFGAVCEPSVEDPGRASCVCKKNACPATVAPVCGSDASTYSNECELQRAQCNQQRRIRLLRQGPCGSRDPCANVTCSFGSTCVPSADGQTASCLCPTTCFGAPDGTVCGSDGVDYPSECQLLSHACASQEHIFKKFNGPCDPCQGSMSDLNHICRVNPRTRHPEMLLRPENCPAQHTPICGDDGVTYENDCVMSRIGATRGLLLQKVRSGQCQTRDQCPETCQFNSVCLSRRGRPHCSCDRVTCDGSYRPVCAQDGHTYNNDCWRQQAECRQQRAIPPKHQGPCDQTPSPCHGVQCAFGAVCTVKNGKAECECQRVCSGIYDPVCGSDGVTYGSVCELESMACTLGREIQVARRGPCDPCGQCRFGSLCEVETGRCVCPSECVESAQPVCGSDGHTYASECELHVHACTHQISLYVASAGHCQTCGEKVCTFGAVCSAGQCVCPRCEHPPPGPVCGSDGVTYLSACELREAACQQQVQIEEAHAGPCEPAECGSGGSGSGEDDECEQELCRQRGGIWDEDSEDGPCVCDFSCQSVPRSPVCGSDGVTYGTECDLKKARCESQQELYVAAQGACRGPTLAPLLPVAFPHCAQTPYGCCQDNFTAAQGVGLAGCPSTCHCNPHGSYSGTCDPATGQCSCRPGVGGLRCDRCEPGFWNFRGIVTDGHSGCTPCSCDPRGAVRDDCEQMTGLCSCRPGVAGPKCGQCPDGQVLGHLGCEADPMTPVTCVEIHCEFGASCVEKAGFAQCICPTLTCPEANSTKVCGSDGVTYGNECQLKAIACRQRLDISTQSLGPCQESVTPGASPTSASMTTPRHILSKTLPFPHNSLPLSPGSTTHDWPTPLPISPHTTVSIPRSTAWPVLTVPPTAAASDVTSLATSIFSESGSANGSGDEELSGDEEASGGGSGGLEPPVGSIVVTHGPPIERASCYNSPLGCCSDGKTPSLDSEGSNCPATKAFQGVLELEGVEGQELFYTPEMADPKSELFGETARSIESTLDDLFRNSDVKKDFWSVRLRELGPGKLVRAIVDVHFDPTTAFQASDVGQALLRQIQVSRPWALAVRRPLQEHVRFLDFDWFPTFFTGAATGTTAAMATARATTVSRLPASSVTPRVYPSHTSRPVGRTTAPPTTRRPPTTATNMDRPRTPGHQQPSKSCDSQPCLHGGTCQDQDSGKGFTCSCTAGRGGSVCEKVQPPSMPAFKGHSFLAFPTLRAYHTLRLALEFRALETEGLLLYNGNARGKDFLALALLDGRVQFRFDTGSGPAVLTSLVPVEPGRWHRLELSRHWRQGTLSVDGETPVVGESPSGTDGLNLDTNLYVGGIPEEQVAMVLDRTSVGVGLKGCIRMLDINNQQLELSDWQRAAVQSSGVGECGDHPCLPNPCHGGALCQALEAGMFLCQCPPGRFGPTCADEKSPCQPNPCHGAAPCRVLSSGGAKCECPLGRSGTFCQTVLETAGSRPFLADFNGFSYLELKGLHTFERDLGEKMALEMVFLARGPSGLLLYNGQKTDGKGDFVSLALHNRHLEFCYDLGKGAAVIRSKEPIALGTWVRVFLERNGRKGALQVGDGPRVLGESPKSRKVPHTMLNLKEPLYIGGAPDFSKLARGAAVSSGFSGVIQLVSLRGHQLLTQEHVLRAVDVSPFADHPCTQALGNPCLNGGSCVPREATYECLCPGGFSGLHCEKGLVEKSVGDLETLAFDGRTYIEYLNAVIESELTNEIPAPETLDSRALFSEKALQSNHFELSLRTEATQGLVLWIGKAAERADYMALAIVDGHLQLSYDLGSQPVVLRSTVKVNTNRWLRIRAHREHREGSLQVGNEAPVTGSSPLGATQLDTDGALWLGGLQKLPVGQALPKAYGTGFVGCLRDVVVGHRQLHLLEDAVTKPELRPCPTP 7FEFDFDAFF89CC31 musk_rat Muscle, skeletal receptor tyrosine protein kinase Musk Muscle-specific tyrosine protein kinase receptor protein protein rat Rattus norvegicus (Rat) Rat MRELVNIPLLQMLTLVAFSGTEKLPKAPVITTPLETVDALVEEVATFMCAVESYPQPEISWTRNKILIKLFDTRYSIRENGQLLTILSVEDSDDGIYCCTANNGVGGAVESCGALQVKMKPKITRPPINVKIIEGLKAVLPCTTMGNPKPSVSWIKGDSALRENSRIAVLESGSLRIHNVQKEDAGQYRCVAKNSLGTAYSKLVKLEVEVFARILRAPESHNVTFGSFVTLRCTAIGMPVPTISWIENGNAVSSGSIQENVKDRVIDSRLQLFITKPGLYTCIATNKHGEKFSTAKAAATVSIAEWSKSQKESKGYCAQYRGEVCDAVLVKDSLVFFNTSYPDPEEAQELLIHTAWNELKAVSPLCRPAAEALLCNHLFQECSPGVLPTPMPICREYCLAVKELFCAKEWLAMEGKTHRGLYRSGMHFLPVPECSKLPSMHQDPTACTRLPYLDYKKENITTFPSITSSKPSVDIPNLPASTSSFAVSPAYSMTVIISIMSCFAVFALLTITTLYCCRRRREWKNKKRESAAVTLTTLPSELLLDRLHPNPMYQRMPLLLNPKLLSLEYPRNNIEYVRDIGEGAFGRVFQARAPGLLPYEPFTMVAVKMLKEEASADMQADFQREAALMAEFDNPNIVKLLGVCAVGKPMCLLFEYMAYGDLNEFLRSMSPHTVCSLSHSDLSTRARVSSPGPPPLSCAEQLCIARQVAAGMAYLSERKFVHRDLATRNCLVGENMVVKIADFGLSRNIYSADYYKADGNDAIPIRWMPPESIFYNRYTTESDVWAYGVVLWEIFSYGLQPYYGMAHEEVIYYVRDGNILACPENCPLELYNLMRLCWSKLPADRPSFCSIHRILQRMCERAEGTVGV C146B4E74EE25B24 lrp4_mouse Low-density lipoprotein receptor-related protein 4 Lrp4 Kiaa0816 LDLR dan protein protein mouse Mus musculus Mouse MRRWWGALLLGALLCAHGIASSLECACGRSHFTCAVSALGECTCIPAQWQCDGDNDCGDHSDEDGCTLPTCSPLDFHCDNGKCIRRSWVCDGDNDCEDDSDEQDCPPRECEEDEFPCQNGYCIRSLWHCDGDNDCGDNSDEQCDMRKCSDKEFRCSDGSCIAEHWYCDGDTDCKDGSDEESCPSAVPSPPCNLEEFQCAYGRCILDIYHCDGDDDCGDWSDESDCSSHQPCRSGEFMCDSGLCINSGWRCDGDADCDDQSDERNCTTSMCTAEQFRCRSGRCVRLSWRCDGEDDCADNSDEENCENTGSPQCASDQFLCWNGRCIGQRKLCNGINDCGDSSDESPQQNCRPRTGEENCNVNNGGCAQKCQMVRGAVQCTCHTGYRLTEDGRTCQDVNECAEEGYCSQGCTNTEGAFQCWCEAGYELRPDRRSCKALGPEPVLLFANRIDIRQVLPHRSEYTLLLNNLENAIALDFHHRRELVFWSDVTLDRILRANLNGSNVEEVVSTGLESPGGLAVDWVHDKLYWTDSGTSRIEVANLDGAHRKVLLWQSLEKPRAIALHPMEGTIYWTDWGNTPRIEASSMDGSGRRIIADTHLFWPNGLTIDYAGRRMYWVDAKHHVIERANLDGSHRKAVISQGLPHPFAITVFEDSLYWTDWHTKSINSANKFTGKNQEIIRNKLHFPMDIHTLHPQRQPAGKNRCGDNNGGCTHLCLPSGQNYTCACPTGFRKINSHACAQSLDKFLLFARRMDIRRISFDTEDLSDDVIPLADVRSAVALDWDSRDDHVYWTDVSTDTISRAKWDGTGQEVVVDTSLESPAGLAIDWVTNKLYWTDAGTDRIEVANTDGSMRTVLIWENLDRPRDIVVEPMGGYMYWTDWGASPKIERAGMDASSRQVIISSNLTWPNGLAIDYGSQRLYWADAGMKTIEFAGLDGSKRKVLIGSQLPHPFGLTLYGQRIYWTDWQTKSIQSADRLTGLDRETLQENLENLMDIHVFHRQRPPVTTLCAVENGGCSHLCLRSPNPSGFSCTCPTGINLLRDGKTCSPGMNSFLIFARRIDVRMVSLDIPYFADVVVPINMTMKNTIAIGVDPLEGKVYWSDSTLHRISRASLDGSQHEDIITTGLQTTDGLAVDAIGRKVYWTDTGTNRIEVGNLDGSMRKVLVWQNLDSPRAIVLYHEMGFMYWTDWGENAKLERSGMDGSDRTVLINNNLGWPNGLTVDKTSSQLLWADAHTERIEVADLNGANRHTLVSPVQHPYGLTLLDSYIYWTDWQTRSIHRADKSTGSNVILVRSNLPGLMDIQAVDRAQPLGFNKCGSRNGGCSHLCLPRPSGFSCACPTGIQLKGDRKTCDPSPETYLLFSSRGSIRRISLDTDDHTDVHVPVPGLNNVISLDYDSVHGKVYYTDVFLDVIRRADLNGSNMETVIGHGLKTTDGLAVDWVARNLYWTDTGRNTIEASRLDGSCRKVLINNSLDEPRAIAVFPRKGYLFWTDWGHIAKIERANLDGSERKVLINTDLGWPNGLTLDYDTRRIYWVDAHLDRIESADLNGKLRQVLVSHVSHPFALTQQDRWIYWTDWQTKSIQRVDKYSGRNKETVLANVEGLMDIIVVSPQRQTGTNACGVNNGGCTHLCFARASDFVCACPDEPDGHPCSLVPGLVPPAPRATSMNEKSPVLPNTLPTTLHSSTTKTRTSLEGAGGRCSERDAQLGLCAHSNEAVPAAPGEGLHVSYAIGGLLSILLILLVIAALMLYRHRKSKFTDPGMGNLTYSNPSYRTSTQEVKLEAAPKPAVYNQLCYKKEGGPDHSYTKEKIKIVEGIRLLAGDDAEWGDLKQLRSSRGGLLRDHVCMKTDTVSIQASSGSLDDTETEQLLQEEQSECSSVHTAATPERRGSLPDTGWKHERKLSSESQV 282CA859B76D9BAD agrn-lrp4-1 1 6 unspecified role unspecified role prey prey n-terminal 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 c-terminal mcherry rfp tag red fluorescent protein tag red fluorescent protein RFP red fluorescent protein RFP rfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range mouse-ba_f3 Mouse pro B cells IL-3 dependent mouse-baf3 EBI-6922762 ba_f3 Mouse pro B cells IL-3 dependent BA/F3 baf3 4 unspecified role unspecified role bait bait c-terminal 50kDa fragment sufficient to bind sufficient binding region sufficient to bind c-term range C-terminal range c-term range c-term range C-terminal range c-term range n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection physical association physical association Lrp4 Is a Receptor for Agrin Fig. 2 The information supplied did not allow the unambiguous assignment of this protein to a single UniProt isoform. agrn-lrp4-2 2 4 unspecified role unspecified role bait bait n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range c-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis c-term range C-terminal range c-term range c-term range C-terminal range c-term range n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range region cy5 label cy5 label undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined spofr-sf_21 Spodoptera frugiperda insect cells sf_21 Insect cells derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae. full name: IPLB-SF-21-AE cells are susceptible to Baculovirus infection. 6 unspecified role unspecified role prey prey n-terminal 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 mouse-ba_f3 Mouse pro B cells IL-3 dependent mouse-baf3 EBI-6922762 ba_f3 Mouse pro B cells IL-3 dependent BA/F3 baf3 physical association physical association Cy5-Agrin B8 bound selectively, in a saturable and dose-dependent manner, to BaF3 cells that expressed Lrp4 (Figure 3C). Fig. 3 Half maximal binding at 6 nM Agrin Calcium, increase binding by 20-fold Agrin construct was agrin B8 - a neuronal isoform containing a C-terminal amino acid insert. This could not be unambiguously mapped to the existing UniProt isoforms. It is unclear how the Cy5 label is bound and which of the tags present on the original construct are present after this process. agrn-lrp4-3 2 5 stimulator stimulator prey prey c-terminal 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 n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range mouse-ba_f3 Mouse pro B cells IL-3 dependent mouse-baf3 EBI-6922762 ba_f3 Mouse pro B cells IL-3 dependent BA/F3 baf3 6 unspecified role unspecified role prey prey n-terminal 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 mouse-ba_f3 Mouse pro B cells IL-3 dependent mouse-baf3 EBI-6922762 ba_f3 Mouse pro B cells IL-3 dependent BA/F3 baf3 4 unspecified role unspecified role bait bait c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined c-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis c-term range C-terminal range c-term range c-term range C-terminal range c-term range n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range region cy5 label cy5 label undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range spofr-sf_21 Spodoptera frugiperda insect cells sf_21 Insect cells derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae. full name: IPLB-SF-21-AE cells are susceptible to Baculovirus infection. physical association physical association Consistent with this idea, cells expressing Lrp4 and MuSK bound more Cy5-Agrin than did cells expressing Lrp4 alone, but only at high concentrations of Agrin (Figure 3D). Fig. 3D Agrin construct was agrin B8 - a neuronal isoform containing a C-terminal amino acid insert. This could not be unambiguously mapped to the existing UniProt isoforms. It is unclear how the Cy5 label is bound and which of the tags present on the original construct are present after this process. Cells expressing both Lrp4 and Musk bound Agrin more effectively than cells expressing Lrp4 alone. musk-lrp4-1 3 6 unspecified role unspecified role bait bait c-terminal mcherry rfp tag red fluorescent protein tag red fluorescent protein RFP red fluorescent protein RFP rfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 5 stimulator stimulator prey prey c-terminal 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 n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range mouse-ba_f3 Mouse pro B cells IL-3 dependent mouse-baf3 EBI-6922762 ba_f3 Mouse pro B cells IL-3 dependent BA/F3 baf3 physical association physical association Lrp4 and MuSK interact in cis, as they coimmunoprecipitate when expressed together in the same cell. Fig. 4C Lrp4 and MuSK interaction was shown to be cis i.e. within the same cell. When expressed in different cells, the interaction was poor.