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/ The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn. The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn. J. Biol. Chem. (0021-9258) 2007 imex curation Yun HM., Kim S., Kim HJ., Kostenis E., Kim JI., Seong JY., Baik JH., Rhim H. hrhim@kist.re.kr Human brain cDNA Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid predetermined predetermined participant predetermined Yun HM., Kim S., Kim HJ., Kostenis E., Kim JI., Seong JY., Baik JH., Rhim H. hrhim@kist.re.kr Accepted 2007-JAN-23 by JYOTI. Human brain cDNA J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn. The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn. J. Biol. Chem. (0021-9258) 2007 imex curation Yun HM., Kim S., Kim HJ., Kostenis E., Kim JI., Seong JY., Baik JH., Rhim H. hrhim@kist.re.kr Human brain cDNA Only protein-protein interactions crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Yun HM., Kim S., Kim HJ., Kostenis E., Kim JI., Seong JY., Baik JH., Rhim H. hrhim@kist.re.kr Accepted 2007-JAN-23 by JYOTI. Human brain cDNA J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn. The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn. J. Biol. Chem. (0021-9258) 2007 imex curation Yun HM., Kim S., Kim HJ., Kostenis E., Kim JI., Seong JY., Baik JH., Rhim H. hrhim@kist.re.kr Human brain cDNA Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Yun HM., Kim S., Kim HJ., Kostenis E., Kim JI., Seong JY., Baik JH., Rhim H. hrhim@kist.re.kr Accepted 2007-JAN-23 by JYOTI. Human brain cDNA J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn. The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn. J. Biol. Chem. (0021-9258) 2007 imex curation Yun HM., Kim S., Kim HJ., Kostenis E., Kim JI., Seong JY., Baik JH., Rhim H. hrhim@kist.re.kr Human brain cDNA Only protein-protein interactions crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Yun HM., Kim S., Kim HJ., Kostenis E., Kim JI., Seong JY., Baik JH., Rhim H. hrhim@kist.re.kr Accepted 2007-JAN-23 by JYOTI. Human brain cDNA J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn. The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn. J. Biol. Chem. (0021-9258) 2007 imex curation Yun HM., Kim S., Kim HJ., Kostenis E., Kim JI., Seong JY., Baik JH., Rhim H. hrhim@kist.re.kr Human brain cDNA Only protein-protein interactions human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Yun HM., Kim S., Kim HJ., Kostenis E., Kim JI., Seong JY., Baik JH., Rhim H. hrhim@kist.re.kr Accepted 2007-JAN-23 by JYOTI. Human brain cDNA J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn. The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn. J. Biol. Chem. (0021-9258) 2007 imex curation Yun HM., Kim S., Kim HJ., Kostenis E., Kim JI., Seong JY., Baik JH., Rhim H. hrhim@kist.re.kr Human brain cDNA Only protein-protein interactions in vitro In vitro pull down pull down nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Yun HM., Kim S., Kim HJ., Kostenis E., Kim JI., Seong JY., Baik JH., Rhim H. hrhim@kist.re.kr Accepted 2007-JAN-23 by JYOTI. Human brain cDNA J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn. The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn. J. Biol. Chem. (0021-9258) 2007 imex curation Yun HM., Kim S., Kim HJ., Kostenis E., Kim JI., Seong JY., Baik JH., Rhim H. hrhim@kist.re.kr Human brain cDNA Only protein-protein interactions human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Yun HM., Kim S., Kim HJ., Kostenis E., Kim JI., Seong JY., Baik JH., Rhim H. hrhim@kist.re.kr Accepted 2007-JAN-23 by JYOTI. Human brain cDNA J. Biol. Chem. (0021-9258) 2007 Only protein-protein interactions imex curation g3hmr9_crigr I79_012041 protein protein crigr Cricetulus griseus (Chinese hamster) Cricetulus barabensis griseus Chinese hamster MSPESFLEEAQIMKKLKHDKLVQLYAVVSEEPIYIVTEYMNKGSLLDFLKDGEGRALKLPNLVDMAAQVAAGMAYIERMNYIHRDLRSANILVGNGLICKIADFGLARLIEDNEYTARQGAKFPIKWTAPEAALYGRFTIKSDVWSFGILLTELVTKGRVPYPGMNNREVLEQVERGYRMPCPQDCPISLHELMIHCWKKDPEERPTFEYLQGFLEDYFTATEPQYQPGENL 12293B796FA08AB8 fyn_human Tyrosine-protein kinase Fyn p59-Fyn Proto-oncogene c-Fyn Src-like kinase FYN Proto-oncogene Syn protein protein human Homo sapiens Human MGCVQCKDKEATKLTEERDGSLNQSSGYRYGTDPTPQHYPSFGVTSIPNYNNFHAAGGQGLTVFGGVNSSSHTGTLRTRGGTGVTLFVALYDYEARTEDDLSFHKGEKFQILNSSEGDWWEARSLTTGETGYIPSNYVAPVDSIQAEEWYFGKLGRKDAERQLLSFGNPRGTFLIRESETTKGAYSLSIRDWDDMKGDHVKHYKIRKLDNGGYYITTRAQFETLQQLVQHYSERAAGLCCRLVVPCHKGMPRLTDLSVKTKDVWEIPRESLQLIKRLGNGQFGEVWMGTWNGNTKVAIKTLKPGTMSPESFLEEAQIMKKLKHDKLVQLYAVVSEEPIYIVTEYMNKGSLLDFLKDGEGRALKLPNLVDMAAQVAAGMAYIERMNYIHRDLRSANILVGNGLICKIADFGLARLIEDNEYTARQGAKFPIKWTAPEAALYGRFTIKSDVWSFGILLTELVTKGRVPYPGMNNREVLEQVERGYRMPCPQDCPISLHELMIHCWKKDPEERPTFEYLQSFLEDYFTATEPQYQPGENL DIP protein P06241 original sequence version: 195 DIP protein P06241 original sequence version: 147 DIP protein P06241 original sequence version: 164 4A1E443A4B5A0977 5ht6r_human 5-hydroxytryptamine receptor 6 Serotonin receptor 6 HTR6 protein protein human Homo sapiens Human MVPEPGPTANSTPAWGAGPPSAPGGSGWVAAALCVVIALTAAANSLLIALICTQPALRNTSNFFLVSLFTSDLMVGLVVMPPAMLNALYGRWVLARGLCLLWTAFDVMCCSASILNLCLISLDRYLLILSPLRYKLRMTPLRALALVLGAWSLAALASFLPLLLGWHELGHARPPVPGQCRLLASLPFVLVASGLTFFLPSGAICFTYCRILLAARKQAVQVASLTTGMASQASETLQVPRTPRPGVESADSRRLATKHSRKALKASLTLGILLGMFFVTWLPFFVANIVQAVCDCISPGLFDVLTWLGYCNSTMNPIIYPLFMRDFKRALGRFLPCPRCPRERQASLASPSLRTSHSGPRPGLSLQQVLPLPLPPDSDSDSDAGSGGSSGLRLTAQLLLPGEATQDPPLPTRAAAAVNFFNIDPAEPELRPHPLGIPTN C888F47650C1D2EF fyn-htr6-1 3 10 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 9 unspecified role unspecified role prey prey physical association physical association After screening human brain cDNA library, we identified that Fyn tyrosine kinase interacted with the CT of 5- HT6R. fyn-htr6-2 1 9 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association To confirm this specific interaction, we carried out a yeast two-hybrid assay using CT cDNA (bait) transformed in the AH109 yeast strain and full-length Fyn cDNA (prey) transformed into the Y187 yeast strain as shown Fig. 1A. 1 fyn-htr6-3 6 9 unspecified role unspecified role prey prey region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined sh3 region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 24 22 direct interaction direct interaction Purified His-CT protein interacted with GSTSH3, but not GST or GST-SH2 (Fig. 2B). 2B fyn-htr6-4 7 9 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association As shown in Fig. 2C, the transfected HA-5-HT6R was able to bind to endogenous Fyn (lane 2) in the HEK293 cells while no signal was detected from non-transfected cells (lane 1).. 2C fyn-htr6-5 5 9 unspecified role unspecified role bait bait 10 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association When coimmunoprecipitation was performed in reverse with anti-Fyn antibodies, followed by immunoblotting with anti-HA antibodies, the result was the same (Fig. 2C, lane 3). 2C fyn-htr6-6 4 10 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 9 unspecified role unspecified role prey prey physical association physical association After HA-5-HT6R and Fyn were transfected into CHO/K-1 cells, the immunoprecipitation was performed with either anti-HA or anti-Fyn antibodies. 2D fyn-htr6-7 2 10 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 9 unspecified role unspecified role bait bait physical association physical association When coimmunoprecipitation was performed in reverse with anti-Fyn antibodies, followed by immunoblotting with anti-HA antibodies, the result was the same (Fig. 2C, lane 3). 2C fyn-htr6-8 4 8 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association As shown in Fig. 2D, HA-5-HT6R was co-immunoprecipitated with both exogenous (lane 2 & 3) and endogenous Fyn (lane 4) while no signal was detected in immunoprecipitates from non-transfected cells (lane 1). 2D