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/ Cocaine Blocks Effects of Hunger Hormone, Ghrelin, Via Interaction with Neuronal Sigma-1 Receptors. Cocaine Blocks Effects of Hunger Hormone, Ghrelin, Via Interaction with Neuronal Sigma-1 Receptors. Molecular neurobiology 2019 imex curation Aguinaga D., Medrano M., Cordomí A., Jiménez-Rosés M., Angelats E., Casanovas M., Vega-Quiroga I., Canela EI., Petrovic M., Gysling K., Pardo L., Franco R., Navarro G. Results from PLA experiments (Figs 5+6) have not been included due to ambiguity in the antibodies used. (anti-Ghrelin or anti -Ghrelin Receptor.) email sent to authors requesting clarification of antibody used for PLA - and also unfused species in fig 2f/g BIFC. (no reply -Franco has responded to another/other emails.) Please confirm - or otherwise- various comments/discrepancies I have mentioned in comments/cautions for this publication. I have been referring to several different versions of the publication which all differ from each other, and it is possible some of these "problems" have been removed. [I have now received the "Kosher" copy of all files from the library including Supp. Table 1 with peptide sequences;however discrepancies have NOT been resolved.] Please email me if you need any of the files and I will forward stuff from library. rfranco123@gmail.com,rfranco@ub.edu,dimartts@hotmail.com The email address for Gemma Navarro does not work; nor does g.navarro@ub.edu 21-Nov-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. confocal microscopy confocal microscopy tag fluorescence tag visualisation by fluorescence tag fluorescence Aguinaga D., Medrano M., Cordomí A., Jiménez-Rosés M., Angelats E., Casanovas M., Vega-Quiroga I., Canela EI., Petrovic M., Gysling K., Pardo L., Franco R., Navarro G. Results from PLA experiments (Figs 5+6) have not been included due to ambiguity in the antibodies used. (anti-Ghrelin or anti -Ghrelin Receptor.) email sent to authors requesting clarification of antibody used for PLA - and also unfused species in fig 2f/g BIFC. (no reply -Franco has responded to another/other emails.) rfranco123@gmail.com,rfranco@ub.edu,dimartts@hotmail.com The email address for Gemma Navarro does not work; nor does g.navarro@ub.edu Accepted 2019-OCT-02 AT 13:53 BST AT 13:53 BST by PPORRAS Molecular neurobiology 2019 Only protein-protein interactions imex curation imex curation Cocaine Blocks Effects of Hunger Hormone, Ghrelin, Via Interaction with Neuronal Sigma-1 Receptors. Cocaine Blocks Effects of Hunger Hormone, Ghrelin, Via Interaction with Neuronal Sigma-1 Receptors. Molecular neurobiology 2019 imex curation Aguinaga D., Medrano M., Cordomí A., Jiménez-Rosés M., Angelats E., Casanovas M., Vega-Quiroga I., Canela EI., Petrovic M., Gysling K., Pardo L., Franco R., Navarro G. Results from PLA experiments (Figs 5+6) have not been included due to ambiguity in the antibodies used. (anti-Ghrelin or anti -Ghrelin Receptor.) email sent to authors requesting clarification of antibody used for PLA - and also unfused species in fig 2f/g BIFC. (no reply -Franco has responded to another/other emails.) Please confirm - or otherwise- various comments/discrepancies I have mentioned in comments/cautions for this publication. I have been referring to several different versions of the publication which all differ from each other, and it is possible some of these "problems" have been removed. [I have now received the "Kosher" copy of all files from the library including Supp. Table 1 with peptide sequences;however discrepancies have NOT been resolved.] Please email me if you need any of the files and I will forward stuff from library. rfranco123@gmail.com,rfranco@ub.edu,dimartts@hotmail.com The email address for Gemma Navarro does not work; nor does g.navarro@ub.edu 21-Nov-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. bifc bimolecular fluorescence complementation bifc tag fluorescence tag visualisation by fluorescence tag fluorescence Aguinaga D., Medrano M., Cordomí A., Jiménez-Rosés M., Angelats E., Casanovas M., Vega-Quiroga I., Canela EI., Petrovic M., Gysling K., Pardo L., Franco R., Navarro G. Results from PLA experiments (Figs 5+6) have not been included due to ambiguity in the antibodies used. (anti-Ghrelin or anti -Ghrelin Receptor.) email sent to authors requesting clarification of antibody used for PLA - and also unfused species in fig 2f/g BIFC. (no reply -Franco has responded to another/other emails.) rfranco123@gmail.com,rfranco@ub.edu,dimartts@hotmail.com The email address for Gemma Navarro does not work; nor does g.navarro@ub.edu Accepted 2019-OCT-02 AT 13:53 BST AT 13:53 BST by PPORRAS Molecular neurobiology 2019 Only protein-protein interactions imex curation imex curation Cocaine Blocks Effects of Hunger Hormone, Ghrelin, Via Interaction with Neuronal Sigma-1 Receptors. Cocaine Blocks Effects of Hunger Hormone, Ghrelin, Via Interaction with Neuronal Sigma-1 Receptors. Molecular neurobiology 2019 imex curation Aguinaga D., Medrano M., Cordomí A., Jiménez-Rosés M., Angelats E., Casanovas M., Vega-Quiroga I., Canela EI., Petrovic M., Gysling K., Pardo L., Franco R., Navarro G. Results from PLA experiments (Figs 5+6) have not been included due to ambiguity in the antibodies used. (anti-Ghrelin or anti -Ghrelin Receptor.) email sent to authors requesting clarification of antibody used for PLA - and also unfused species in fig 2f/g BIFC. (no reply -Franco has responded to another/other emails.) Please confirm - or otherwise- various comments/discrepancies I have mentioned in comments/cautions for this publication. I have been referring to several different versions of the publication which all differ from each other, and it is possible some of these "problems" have been removed. [I have now received the "Kosher" copy of all files from the library including Supp. Table 1 with peptide sequences;however discrepancies have NOT been resolved.] Please email me if you need any of the files and I will forward stuff from library. rfranco123@gmail.com,rfranco@ub.edu,dimartts@hotmail.com The email address for Gemma Navarro does not work; nor does g.navarro@ub.edu 21-Nov-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation rat-striatal neurons Rattus norvegicus brain-striatum_neuron striatal neurons pla proximity ligation assay pELISA pELISA PLA antibody detection identification by antibody antibody detection Aguinaga D., Medrano M., Cordomí A., Jiménez-Rosés M., Angelats E., Casanovas M., Vega-Quiroga I., Canela EI., Petrovic M., Gysling K., Pardo L., Franco R., Navarro G. Results from PLA experiments (Figs 5+6) have not been included due to ambiguity in the antibodies used. (anti-Ghrelin or anti -Ghrelin Receptor.) email sent to authors requesting clarification of antibody used for PLA - and also unfused species in fig 2f/g BIFC. (no reply -Franco has responded to another/other emails.) rfranco123@gmail.com,rfranco@ub.edu,dimartts@hotmail.com The email address for Gemma Navarro does not work; nor does g.navarro@ub.edu Accepted 2019-OCT-02 AT 13:53 BST AT 13:53 BST by PPORRAS Molecular neurobiology 2019 Only protein-protein interactions imex curation imex curation Cocaine Blocks Effects of Hunger Hormone, Ghrelin, Via Interaction with Neuronal Sigma-1 Receptors. Cocaine Blocks Effects of Hunger Hormone, Ghrelin, Via Interaction with Neuronal Sigma-1 Receptors. Molecular neurobiology 2019 imex curation Aguinaga D., Medrano M., Cordomí A., Jiménez-Rosés M., Angelats E., Casanovas M., Vega-Quiroga I., Canela EI., Petrovic M., Gysling K., Pardo L., Franco R., Navarro G. Results from PLA experiments (Figs 5+6) have not been included due to ambiguity in the antibodies used. (anti-Ghrelin or anti -Ghrelin Receptor.) email sent to authors requesting clarification of antibody used for PLA - and also unfused species in fig 2f/g BIFC. (no reply -Franco has responded to another/other emails.) Please confirm - or otherwise- various comments/discrepancies I have mentioned in comments/cautions for this publication. I have been referring to several different versions of the publication which all differ from each other, and it is possible some of these "problems" have been removed. [I have now received the "Kosher" copy of all files from the library including Supp. Table 1 with peptide sequences;however discrepancies have NOT been resolved.] Please email me if you need any of the files and I will forward stuff from library. rfranco123@gmail.com,rfranco@ub.edu,dimartts@hotmail.com The email address for Gemma Navarro does not work; nor does g.navarro@ub.edu 21-Nov-2019: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. bret bioluminescence resonance energy transfer BRET LRET LRET BRET bret tag visualisation tag visualisation Aguinaga D., Medrano M., Cordomí A., Jiménez-Rosés M., Angelats E., Casanovas M., Vega-Quiroga I., Canela EI., Petrovic M., Gysling K., Pardo L., Franco R., Navarro G. Results from PLA experiments (Figs 5+6) have not been included due to ambiguity in the antibodies used. (anti-Ghrelin or anti -Ghrelin Receptor.) email sent to authors requesting clarification of antibody used for PLA - and also unfused species in fig 2f/g BIFC. (no reply -Franco has responded to another/other emails.) rfranco123@gmail.com,rfranco@ub.edu,dimartts@hotmail.com The email address for Gemma Navarro does not work; nor does g.navarro@ub.edu Accepted 2019-OCT-02 AT 13:53 BST AT 13:53 BST by PPORRAS Molecular neurobiology 2019 Only protein-protein interactions imex curation imex curation sgmr1_human Sigma non-opioid intracellular receptor 1 SIGMAR1 OPRS1 SRBP Aging-associated gene 8 protein SR31747-binding protein Sigma 1-type opioid receptor AAG8 protein protein human Homo sapiens Human MQWAVGRRWAWAALLLAVAAVLTQVVWLWLGTQSFVFQREEIAQLARQYAGLDHELAFSRLIVELRRLHPGHVLPDEELQWVFVNAGGWMGAMCLLHASLSEYVLLFGTALGSRGHSGRYWAEISDTIISGTFHQWREGTTKSEVFYPGETVVHGPGEATAVEWGPNTWMVEYGRGVIPSTLAFALADTVFSTQDFLTLFYTLRSYARGLRLELTTYLFGQDP 0C498636BEB1C443 ghrl_rat Appetite-regulating hormone Growth hormone secretagogue Growth hormone-releasing peptide Motilin-related peptide Ghrl protein protein rat Rattus norvegicus (Rat) Rat MVSSATICSLLLLSMLWMDMAMAGSSFLSPEHQKAQQRKESKKPPAKLQPRALEGWLHPEDRGQAEEAEEELEIRFNAPFDVGIKLSGAQYQQHGRALGKFLQDILWEEVKEAPANK 8857546FE51A7691 q92847-2 Growth hormone secretagogue receptor type 1 GH-releasing peptide receptor Ghrelin receptor GHSR protein protein human Homo sapiens Human MWNATPSEEPGFNLTLADLDWDASPGNDSLGDELLQLFPAPLLAGVTATCVALFVVGIAGNLLTMLVVSRFRELRTTTNLYLSSMAFSDLLIFLCMPLDLVRLWQYRPWNFGDLLCKLFQFVSESCTYATVLTITALSVERYFAICFPLRAKVVVTKGRVKLVIFVIWAVAFCSAGPIFVLVGVEHENGTDPWDTNECRPTEFAVRSGLLTVMVWVSSIFFFLPVFCLTVLYSLIGRKLWRRRRGDAVVGASLRDQNHKQTVKMLGGSQRALRLSLAGPILSLCLLPSL 8032FDB4C6BDD42B sgmr1_rat Sigma non-opioid intracellular receptor 1 Sigma 1-type opioid receptor Sigmar1 Oprs1 protein protein rat Rattus norvegicus (Rat) Rat MPWAVGRRWAWITLFLTIVAVLIQAVWLWLGTQSFVFQREEIAQLARQYAGLDHELAFSRLIVELRRLHPGHVLPDEELQWVFVNAGGWMGAMCLLHASLSEYVLLFGTALGSHGHSGRYWAEISDTIISGTFHQWREGTTKSEVYYPGETVVHGPGEATAVEWGPNTWMVEYGRGVIPSTLAFALSDTIFSTQDFLTLFYTLRAYARGLRLELTTYLFGQDP B125A0388F1FFC6E ghsr_rat Growth hormone secretagogue receptor type 1 GH-releasing peptide receptor Ghrelin receptor Ghsr protein protein rat Rattus norvegicus (Rat) Rat MWNATPSEEPEPNVTLDLDWDASPGNDSLPDELLPLFPAPLLAGVTATCVALFVVGISGNLLTMLVVSRFRELRTTTNLYLSSMAFSDLLIFLCMPLDLVRLWQYRPWNFGDLLCKLFQFVSESCTYATVLTITALSVERYFAICFPLRAKVVVTKGRVKLVILVIWAVAFCSAGPIFVLVGVEHENGTDPRDTNECRATEFAVRSGLLTVMVWVSSVFFFLPVFCLTVLYSLIGRKLWRRRGDAAVGASLRDQNHKQTVKMLAVVVFAFILCWLPFHVGRYLFSKSFEPGSLEIAQISQYCNLVSFVLFYLSAAINPILYNIMSKKYRVAVFKLLGFESFSQRKLSTLKDESSRAWTKSSINT DCBF559BE061EEE9 q92847-1 Growth hormone secretagogue receptor type 1 GH-releasing peptide receptor Ghrelin receptor GHSR protein protein human Homo sapiens Human MWNATPSEEPGFNLTLADLDWDASPGNDSLGDELLQLFPAPLLAGVTATCVALFVVGIAGNLLTMLVVSRFRELRTTTNLYLSSMAFSDLLIFLCMPLDLVRLWQYRPWNFGDLLCKLFQFVSESCTYATVLTITALSVERYFAICFPLRAKVVVTKGRVKLVIFVIWAVAFCSAGPIFVLVGVEHENGTDPWDTNECRPTEFAVRSGLLTVMVWVSSIFFFLPVFCLTVLYSLIGRKLWRRRRGDAVVGASLRDQNHKQTVKMLAVVVFAFILCWLPFHVGRYLFSKSFEPGSLEIAQISQYCNLVSFVLFYLSAAINPILYNIMSKKYRVAVFRLLGFEPFSQRKLSTLKDESSRAWTESSINT D1B62710DA9DC0C6 ghsr-sigmar1-1 1 10 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 c-terminus renilla luciferase renilla luciferase protein tag renilla luciferase c-term range C-terminal range c-term range c-term range C-terminal range c-term range 5 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization Figure 1A,3A cocaine PRE-084 -a Sigma 1R agonist Both Cocaine and PRE-084 increased colocalization of sigma- 1 R and GHS-R1a at the cell surface. ghsr-sigmar1-2 4 10 immunocytochemistry immunocytochemistry tag visualisation tag visualisation unspecified role unspecified role luminescence accept luminescence acceptor luminescence 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 5 unspecified role unspecified role luminescence donor luminescence donor over-expressed over expressed level over-expressed c-terminus renilla luciferase renilla luciferase protein tag renilla luciferase c-term range C-terminal range c-term range c-term range C-terminal range c-term range proximity proximity close-contact colocalization neighbourhood interaction Figure 1C, 3B Addition of Cocaine or PRE-084 (Sigma-1R agonist) did not show any significant effect of treatments versus control. ghsr-ghsr-1 2 10 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region cyfp cYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region nyfp nYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 2A-Control In the presence of interference peptides, fluorescence decreased by twofold only with TM5 and TM6 peptides. ghsr-ghsr-2 2 7 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region cyfp cYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region nyfp nYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 2B/C-Control In the presence of interference peptides, fluorescence decreased by twofold only with TM5 and TM6 peptides. However in the presence of non-fused GHS-R1a, fluorescence was reduced in the presence of TM4, TM5, and TM6 peptides. ghsr-ghsr-3 2 5 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region cyfp cYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region nyfp nYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 2E-control In the presence of interference peptides, fluorescence decreased with TM1, TM2, and TM5 peptides. ghsr-sigmar1-3 2 5 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region cyfp cYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region nyfp nYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 2F/G control In the presence of interference peptides, fluorescence decreased only with TM1 and TM2 peptides. A similar result was obtained in the presence of unfused GHS-R1b. ghsr-sigmar1-4 3 6 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level 8 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level 9 unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level proximity proximity close-contact colocalization neighbourhood interaction Figure 5C/D, 6 It is stated that "GHS-R1a-Sigma 1 R heteroreceptor complexes were identified in striatal primary cultures". The Methods section states use of "anti-GHS-R1a antibody (1/100, AbCam,Cambridge, UK)";however the image legend text states that "a polyclonal anti-ghrelin primary antibody (1/100, AbCam," was used...). The only Uniprot entry for Rat GHSR has been used here as that would appear to be the most probable protein detected. Cocaine [This PLA experiment probably should be deleted: left in to get another opinion. They state in some instances an "anti-ghrelin antibody was used, other instances aantibody to the ghrelin Receptor. Abcam do not sell a specific "anti-GHS-R1a antibody "., and Uniprot (+NCBI) do not list any isoforms for the RAT protein ]. So we do not know what is being detected. I have left a comment at Publication level to the effect PLA results have not been included. I am unclear what the difference between Fig5C/D and Fig 6 is: 5C/D might be cells (neurons) and 6c tissue sections - but methods section does not describe separately/distinguish. Interaction was seen in thick striatal sections and also striatal primary cultures (neurons). ghsr-ghsr-4 2 10 competitor competitor competitor unspecified role unspecified role purified purified TM6_peptide sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain n-terminus tat tag transactivating tag Tat tag tat tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range chemical synthesis Chemical synthesis (Chemical synthesis) 10 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region cyfp cYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region nyfp nYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 2A-2 In the presence of interference peptides, fluorescence decreased by twofold only with TM5 and TM6 peptides. ghsr-ghsr-5 2 10 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region cyfp cYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region nyfp nYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 competitor competitor competitor unspecified role unspecified role purified purified TM5_peptide sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain c-terminus tat tag transactivating tag Tat tag tat tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association Figure 2A-1 In the presence of interference peptides, fluorescence decreased by twofold only with TM5 and TM6 peptides. ghsr-ghsr-6 2 10 competitor competitor competitor unspecified role unspecified role purified purified TM5_peptide sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain c-terminus tat tag transactivating tag Tat tag tat tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range chemical synthesis Chemical synthesis (Chemical synthesis) 7 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region cyfp cYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region nyfp nYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 2B/C-1 In the presence of interference peptides, fluorescence decreased by twofold only with TM5 and TM6 peptides. However in the presence of non-fused GHS-R1a, fluorescence was reduced in the presence of TM4, TM5, and TM6 peptides. ghsr-ghsr-7 2 10 competitor competitor competitor unspecified role unspecified role purified purified TM6_peptide sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain n-terminus tat tag transactivating tag Tat tag tat tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range chemical synthesis Chemical synthesis (Chemical synthesis) 7 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region cyfp cYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region nyfp nYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 2B/C-2 In the presence of interference peptides, fluorescence decreased by twofold only with TM5 and TM6 peptides. However in the presence of non-fused GHS-R1a, fluorescence was reduced in the presence of TM4, TM5, and TM6 peptides. ghsr-sigmar1-5 2 10 competitor competitor competitor unspecified role unspecified role purified purified TM5_peptide sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain c-terminus tat tag transactivating tag Tat tag tat tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range chemical synthesis Chemical synthesis (Chemical synthesis) 5 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region cyfp cYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region nyfp nYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 2E-1 In the presence of interference peptides, fluorescence decreased with TM1, TM2, and TM5 peptides. ghsr-ghsr-8 2 10 competitor competitor competitor unspecified role unspecified role purified purified TM1_peptide sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain c-terminus tat tag transactivating tag Tat tag tat tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range chemical synthesis Chemical synthesis (Chemical synthesis) 5 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region cyfp cYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region nyfp nYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 2E-2 In the presence of interference peptides, fluorescence decreased with TM1, TM2, and TM5 peptides. ghsr-ghsr-9 2 10 competitor competitor competitor unspecified role unspecified role purified purified TM2_peptide sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain n-terminus tat tag transactivating tag Tat tag tat tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range chemical synthesis Chemical synthesis (Chemical synthesis) 5 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region cyfp cYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region nyfp nYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 2E-3 In the presence of interference peptides, fluorescence decreased with TM1, TM2, and TM5 peptides. ghsr-sigmar1-6 2 10 competitor competitor competitor unspecified role unspecified role purified purified TM1_peptide sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain c-terminus tat tag transactivating tag Tat tag tat tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range chemical synthesis Chemical synthesis (Chemical synthesis) 5 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region cyfp cYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region nyfp nYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 2F/G-1 In the presence of interference peptides, fluorescence decreased only with TM1 and TM2 peptides. A similar result was obtained in the presence of unfused GHS-R1b. ghsr-ghsr-10 2 10 competitor competitor competitor unspecified role unspecified role purified purified TM2_peptide sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain n-terminus tat tag transactivating tag Tat tag tat tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range chemical synthesis Chemical synthesis (Chemical synthesis) 5 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region cyfp cYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region nyfp nYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 2F/G-2 In the presence of interference peptides, fluorescence decreased only with TM1 and TM2 peptides. A similar result was obtained in the presence of unfused GHS-R1b. ghsr-ghsr-17 2 10 competitor competitor competitor unspecified role unspecified role purified purified TM4_peptide sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain n-terminus tat tag transactivating tag Tat tag tat tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range chemical synthesis Chemical synthesis (Chemical synthesis) 7 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region cyfp cYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed region nyfp nYFP undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Figure 2B/C-3 In the presence of interference peptides, fluorescence decreased by twofold only with TM5 and TM6 peptides. However in the presence of non-fused GHS-R1a, fluorescence was reduced in the presence of TM4, TM5, and TM6 peptides.