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/ ortiz-2005-2 yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast lexa b52 complement lexa b52 complementation LexA B52 transcription complementation lexa b52 complement predetermined predetermined participant predetermined Ortiz JA., Castillo M., del Toro ED., Mulet J., Gerber S., Valor LM., Sala S., Sala F., Gutiérrez LM., Criado M. manuel.criado@umh.es. human adult brain Journal of neurochemistry 2005 Only protein-protein interactions imex curation imex curation ortiz-2005-1 yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast lexa b52 complement lexa b52 complementation LexA B52 transcription complementation lexa b52 complement nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Ortiz JA., Castillo M., del Toro ED., Mulet J., Gerber S., Valor LM., Sala S., Sala F., Gutiérrez LM., Criado M. manuel.criado@umh.es. human adult brain Journal of neurochemistry 2005 Only protein-protein interactions imex curation imex curation ortiz-2005-3 in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Ortiz JA., Castillo M., del Toro ED., Mulet J., Gerber S., Valor LM., Sala S., Sala F., Gutiérrez LM., Criado M. manuel.criado@umh.es. Journal of neurochemistry 2005 Only protein-protein interactions imex curation imex curation ortiz-2005-4 chlae-vero Chlorocebus (Cercopithecus) aethiops kidney cells Grivet vero Chlorocebus (Cercopithecus) aethiops kidney cells similar to cos-7 cells but these are not trasnformed confocal microscopy confocal microscopy tag fluorescence tag visualisation by fluorescence tag fluorescence Ortiz JA., Castillo M., del Toro ED., Mulet J., Gerber S., Valor LM., Sala S., Sala F., Gutiérrez LM., Criado M. 2-2 cells:a derivative from VERO cells. manuel.criado@umh.es. Journal of neurochemistry 2005 Only protein-protein interactions imex curation imex curation acha4_human Neuronal acetylcholine receptor subunit alpha-4 CHRNA4 NACRA4 protein protein human Homo sapiens Human MELGGPGAPRLLPPLLLLLGTGLLRASSHVETRAHAEERLLKKLFSGYNKWSRPVANISDVVLVRFGLSIAQLIDVDEKNQMMTTNVWVKQEWHDYKLRWDPADYENVTSIRIPSELIWRPDIVLYNNADGDFAVTHLTKAHLFHDGRVQWTPPAIYKSSCSIDVTFFPFDQQNCTMKFGSWTYDKAKIDLVNMHSRVDQLDFWESGEWVIVDAVGTYNTRKYECCAEIYPDITYAFVIRRLPLFYTINLIIPCLLISCLTVLVFYLPSECGEKITLCISVLLSLTVFLLLITEIIPSTSLVIPLIGEYLLFTMIFVTLSIVITVFVLNVHHRSPRTHTMPTWVRRVFLDIVPRLLLMKRPSVVKDNCRRLIESMHKMASAPRFWPEPEGEPPATSGTQSLHPPSPSFCVPLDVPAEPGPSCKSPSDQLPPQQPLEAEKASPHPSPGPCRPPHGTQAPGLAKARSLSVQHMSSPGEAVEGGVRCRSRSIQYCVPRDDAAPEADGQAAGALASRNTHSAELPPPDQPSPCKCTCKKEPSSVSPSATVKTRSTKAPPPHLPLSPALTRAVEGVQYIADHLKAEDTDFSVKEDWKYVAMVIDRIFLWMFIIVCLLGTVGLFLPPWLAGMI mint homomint After binding acetylcholine, the AChR responds by an extensive change in conformation that affects all subunits and leads to opening of an ion-conducting channel across the plasma membrane Defects in CHRNA4 are the cause of autosomal dominant nocturnal frontal lobe epilepsy type 1 (ENFL1) [MIM:600513]; also symbolized ADNFLE. ENFL1 is a disease characterized by clusters of motor seizures during sleep q6uxh1-1 Protein disulfide isomerase CRELD2 CRELD2 Cysteine-rich with EGF-like domain protein 2 UNQ185/PRO211 Delta protein protein human Homo sapiens Human MRLPRRAALGLLPLLLLLPPAPEAAKKPTPCHRCRGLVDKFNQGMVDTAKKNFGGGNTAWEEKTLSKYESSEIRLLEILEGLCESSDFECNQMLEAQEEHLEAWWLQLKSEYPDLFEWFCVKTLKVCCSPGTYGPDCLACQGGSQRPCSGNGHCSGDGSRQGDGSCRCHMGYQGPLCTDCMDGYFSSLRNETHSICTACDESCKTCSGLTNRDCGECEVGWVLDEGACVDVDECAAEPPPCSAAQFCKNANGSYTCEECDSSCVGCTGEGPGNCKECISGYAREHGQCADVDECSLAEKTCVRKNENCYNTPGSYVCVCPDGFEETEDACVPPAEAEATEGESPTQLPSREDL q6uxh1-3 Protein disulfide isomerase CRELD2 CRELD2 Cysteine-rich with EGF-like domain protein 2 UNQ185/PRO211 Beta protein protein human Homo sapiens Human MRLPRRAALGLLPLLLLLPPAPEAAKKPTPCHRCRGLVDKFNQGMVDTAKKNFGGGNTAWEEKTLSKYESSEIRLLEILEGLCESSDFECNQMLEAQEEHLEAWWLQLKSEYPDLFEWFCVKTLKVCCSPGTYGPDCLACQGGSQRPCSGNGHCSGDGSRQGDGSCRCHMGYQGPLCTDCMDGYFSSLRNETHSICTACDESCKTCSGLTNRDCGECEVGWVLDEGACVDVDECAAEPPPCSAAQFCKNANGSYTCEGGPGGRVCTPGPAGFRCCLCQHSFMAS Minor isoform. achb2_human Neuronal acetylcholine receptor subunit beta-2 CHRNB2 protein protein human Homo sapiens Human MARRCGPVALLLGFGLLRLCSGVWGTDTEERLVEHLLDPSRYNKLIRPATNGSELVTVQLMVSLAQLISVHEREQIMTTNVWLTQEWEDYRLTWKPEEFDNMKKVRLPSKHIWLPDVVLYNNADGMYEVSFYSNAVVSYDGSIFWLPPAIYKSACKIEVKHFPFDQQNCTMKFRSWTYDRTEIDLVLKSEVASLDDFTPSGEWDIVALPGRRNENPDDSTYVDITYDFIIRRKPLFYTINLIIPCVLITSLAILVFYLPSDCGEKMTLCISVLLALTVFLLLISKIVPPTSLDVPLVGKYLMFTMVLVTFSIVTSVCVLNVHHRSPTTHTMAPWVKVVFLEKLPALLFMQQPRHHCARQRLRLRRRQREREGAGALFFREAPGADSCTCFVNRASVQGLAGAFGAEPAPVAGPGRSGEPCGCGLREAVDGVRFIADHMRSEDDDQSVSEDWKYVAMVIDRLFLWIFVFVCVFGTIGMFLQPLFQNYTTTTFLHSDHSAPSSK chrna4-creld2-2 3559283 N/A 3559287 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cytoplasmic_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false N/A 3559288 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false Figure 3C GenBank accession number NP_077300. ("alpha" isoform) A weaker interaction than with CRELD2 - beta isoform. chrna4-creld2-3 3559283 N/A 3559287 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false N/A 3559289 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false Figure 3A/B/C Clone B260, "beta" isoform. (Genbank identifier: AJ968414) chrna4-creld2-4 3559283 N/A 3559289 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false N/A 3559290 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false Figure 3B Clone B260, "beta" isoform. (Genbank identifier: AJ968414) chrna4-1 3559284 N/A 3559287 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed cytoplasmic_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false N/A 3559288 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed physical association physical association false false false no figure GenBank accession number NP_077300. ("alpha" isoform) chrna4-creld2-1 3559284 N/A 3559287 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed cytoplasmic_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false N/A 3559289 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed physical association physical association false false false no figure Clone B260, "beta" isoform. (Genbank identifier: AJ968414) creld2-chrna4-1 3559285 N/A 3559287 unspecified role unspecified role bait bait purified purified cytoplasmic_region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n-terminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) N/A 3559288 unspecified role unspecified role prey prey cell lysate cell lysate region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false rabit-reticulocyte Oryctolagus cuniculus reticulocyte reticulocyte Reticulocytes physical association physical association false false false Figure 4 GenBank accession number NP_077300. ("alpha" isoform) The N-terminal domain of CRELD2 (residues 1–130), common to both a and b isoforms, showed a much weaker interaction with the a4 cytoplasmic loop than the whole protein. A similar result was obtained using CRELD2 C-terminal regions. chrna4-creld2-5 3559285 N/A 3559287 unspecified role unspecified role bait bait purified purified region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n-terminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) N/A 3559289 unspecified role unspecified role prey prey cell lysate cell lysate region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false rabit-reticulocyte Oryctolagus cuniculus reticulocyte reticulocyte Reticulocytes physical association physical association false false false Figure 4 Clone B260, "beta" isoform. (Genbank identifier: AJ968414) The N-terminal domain of CRELD2 (residues 1–130), common to both a and b isoforms, showed a much weaker interaction with the a4 cytoplasmic loop than the whole protein. A similar result was obtained using CRELD2 C-terminal regions. chrnb2-creld2-1 3559286 N/A 3559289 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region rfp tag red fluorescent protein tag red fluorescent protein RFP red fluorescent protein RFP rfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 3559290 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 6 Untagged alpha-4 nAChR (CHRNA4;Uniprot ID = P43681) was also transfected.