Recombinant human melatonin receptor MT1 isolated in mixed detergents shows pharmacology similar to that in mammalian cell membranes
The human melatonin MT1 receptor-belonging to the large family of G protein-coupled receptors (GPCRs)-plays a key role in circadian rhythm regulation and is notably involved in sleep disorders and depression. Structural and functional information at the molecular level are highly desired for fine ch...
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| Veröffentlicht in: | PloS one 2014-06, Vol.9 (6), p.e100616-e100616 |
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| creator | Logez, Christel Berger, Sylvie Legros, Céline Banères, Jean-Louis Cohen, William Delagrange, Philippe Nosjean, Olivier Boutin, Jean A Ferry, Gilles Simonin, Frédéric Wagner, Renaud |
| description | The human melatonin MT1 receptor-belonging to the large family of G protein-coupled receptors (GPCRs)-plays a key role in circadian rhythm regulation and is notably involved in sleep disorders and depression. Structural and functional information at the molecular level are highly desired for fine characterization of this receptor; however, adequate techniques for isolating soluble MT1 material suitable for biochemical and biophysical studies remain lacking. Here we describe the evaluation of a panel of constructs and host systems for the production of recombinant human MT1 receptors, and the screening of different conditions for their solubilization and purification. Our findings resulted in the establishment of an original strategy using a mixture of Fos14 and CHAPS detergents to extract and purify a recombinant human MT1 from Pichia pastoris membranes. This procedure enabled the recovery of relatively pure, monomeric and ligand-binding active MT1 receptor in the near-milligram range. A comparative study based on extensive ligand-binding characterization highlighted a very close correlation between the pharmacological profiles of MT1 purified from yeast and the same receptor present in mammalian cell membranes. The high quality of the purified MT1 was further confirmed by its ability to activate its cognate Gαi protein partner when reconstituted in lipid discs, thus opening novel paths to investigate this receptor by biochemical and biophysical approaches. |
| doi_str_mv | 10.1371/journal.pone.0100616 |
| format | Article |
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Structural and functional information at the molecular level are highly desired for fine characterization of this receptor; however, adequate techniques for isolating soluble MT1 material suitable for biochemical and biophysical studies remain lacking. Here we describe the evaluation of a panel of constructs and host systems for the production of recombinant human MT1 receptors, and the screening of different conditions for their solubilization and purification. Our findings resulted in the establishment of an original strategy using a mixture of Fos14 and CHAPS detergents to extract and purify a recombinant human MT1 from Pichia pastoris membranes. This procedure enabled the recovery of relatively pure, monomeric and ligand-binding active MT1 receptor in the near-milligram range. A comparative study based on extensive ligand-binding characterization highlighted a very close correlation between the pharmacological profiles of MT1 purified from yeast and the same receptor present in mammalian cell membranes. The high quality of the purified MT1 was further confirmed by its ability to activate its cognate Gαi protein partner when reconstituted in lipid discs, thus opening novel paths to investigate this receptor by biochemical and biophysical approaches.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0100616</identifier><identifier>PMID: 24959712</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Animals ; Binding ; Biology and Life Sciences ; Cell Line ; Cell Membrane ; Cell Membrane - chemistry ; Cell Membrane - metabolism ; Cell membranes ; Chemical Sciences ; CHO Cells ; Cholesterol ; Chromatography ; Circadian rhythms ; Comparative studies ; Cricetulus ; Detergents ; Detergents - chemistry ; Escherichia coli ; Escherichia coli - genetics ; Escherichia coli - metabolism ; G protein-coupled receptors ; G proteins ; Gene Expression ; Host systems ; Humans ; Ligands ; Lipids ; Mammals ; Melatonin ; Membranes ; Mental depression ; Pharmacology ; Pichia pastoris ; Protein Binding ; Proteins ; Receptor, Melatonin, MT1 ; Receptor, Melatonin, MT1 - chemistry ; Receptor, Melatonin, MT1 - genetics ; Receptor, Melatonin, MT1 - metabolism ; Receptors ; Recombinant Proteins ; Recombinant Proteins - genetics ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - metabolism ; Sleep ; Sleep disorders ; Solubilization ; Structure-function relationships ; Yeast ; Yeasts ; Yeasts - genetics ; Yeasts - metabolism</subject><ispartof>PloS one, 2014-06, Vol.9 (6), p.e100616-e100616</ispartof><rights>COPYRIGHT 2014 Public Library of Science</rights><rights>2014 Logez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>Attribution</rights><rights>2014 Logez et al 2014 Logez et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c726t-7fdd2d5ed6d5df9263288b258e747597453cd9ecd5c72f0ae77fac6c7a2974993</citedby><cites>FETCH-LOGICAL-c726t-7fdd2d5ed6d5df9263288b258e747597453cd9ecd5c72f0ae77fac6c7a2974993</cites><orcidid>0000-0001-7078-1285</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4069108/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4069108/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79569,79570</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24959712$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://hal.umontpellier.fr/hal-03573074$$DView record in HAL$$Hfree_for_read</backlink></links><search><creatorcontrib>Logez, Christel</creatorcontrib><creatorcontrib>Berger, Sylvie</creatorcontrib><creatorcontrib>Legros, Céline</creatorcontrib><creatorcontrib>Banères, Jean-Louis</creatorcontrib><creatorcontrib>Cohen, William</creatorcontrib><creatorcontrib>Delagrange, Philippe</creatorcontrib><creatorcontrib>Nosjean, Olivier</creatorcontrib><creatorcontrib>Boutin, Jean A</creatorcontrib><creatorcontrib>Ferry, Gilles</creatorcontrib><creatorcontrib>Simonin, Frédéric</creatorcontrib><creatorcontrib>Wagner, Renaud</creatorcontrib><title>Recombinant human melatonin receptor MT1 isolated in mixed detergents shows pharmacology similar to that in mammalian cell membranes</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>The human melatonin MT1 receptor-belonging to the large family of G protein-coupled receptors (GPCRs)-plays a key role in circadian rhythm regulation and is notably involved in sleep disorders and depression. Structural and functional information at the molecular level are highly desired for fine characterization of this receptor; however, adequate techniques for isolating soluble MT1 material suitable for biochemical and biophysical studies remain lacking. Here we describe the evaluation of a panel of constructs and host systems for the production of recombinant human MT1 receptors, and the screening of different conditions for their solubilization and purification. Our findings resulted in the establishment of an original strategy using a mixture of Fos14 and CHAPS detergents to extract and purify a recombinant human MT1 from Pichia pastoris membranes. This procedure enabled the recovery of relatively pure, monomeric and ligand-binding active MT1 receptor in the near-milligram range. A comparative study based on extensive ligand-binding characterization highlighted a very close correlation between the pharmacological profiles of MT1 purified from yeast and the same receptor present in mammalian cell membranes. The high quality of the purified MT1 was further confirmed by its ability to activate its cognate Gαi protein partner when reconstituted in lipid discs, thus opening novel paths to investigate this receptor by biochemical and biophysical approaches.</description><subject>Animals</subject><subject>Binding</subject><subject>Biology and Life Sciences</subject><subject>Cell Line</subject><subject>Cell Membrane</subject><subject>Cell Membrane - chemistry</subject><subject>Cell Membrane - metabolism</subject><subject>Cell membranes</subject><subject>Chemical Sciences</subject><subject>CHO Cells</subject><subject>Cholesterol</subject><subject>Chromatography</subject><subject>Circadian rhythms</subject><subject>Comparative studies</subject><subject>Cricetulus</subject><subject>Detergents</subject><subject>Detergents - chemistry</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - metabolism</subject><subject>G protein-coupled receptors</subject><subject>G proteins</subject><subject>Gene Expression</subject><subject>Host systems</subject><subject>Humans</subject><subject>Ligands</subject><subject>Lipids</subject><subject>Mammals</subject><subject>Melatonin</subject><subject>Membranes</subject><subject>Mental depression</subject><subject>Pharmacology</subject><subject>Pichia pastoris</subject><subject>Protein Binding</subject><subject>Proteins</subject><subject>Receptor, Melatonin, MT1</subject><subject>Receptor, Melatonin, MT1 - chemistry</subject><subject>Receptor, Melatonin, MT1 - genetics</subject><subject>Receptor, Melatonin, MT1 - metabolism</subject><subject>Receptors</subject><subject>Recombinant Proteins</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - metabolism</subject><subject>Sleep</subject><subject>Sleep disorders</subject><subject>Solubilization</subject><subject>Structure-function relationships</subject><subject>Yeast</subject><subject>Yeasts</subject><subject>Yeasts - genetics</subject><subject>Yeasts - metabolism</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNk01v1DAQhiMEoqXwDxBEQkL0sIvtfDi-IFUV0EpFlUrhas3ak42rON7aTmnv_HCc7rbqVj2gHGLNPPOO_dqTZW8pmdOC088XbvQD9POVG3BOKCE1rZ9lu1QUbFYzUjx_sN7JXoVwQUhVNHX9MtthpagEp2w3-3uGytmFGWCIeTdaGHKLPUQ3mCH3qHAVnc9_nNPcBJfiqPOUsOY6LTRG9EscYshD5_6EfNWBt6Bc75Y3eTDW9ODz6PLYQbwtA2uhN6mHwr5PjezCw4DhdfaihT7gm81_L_v17ev54dHs5PT78eHByUxxVscZb7VmukJd60q3gtUFa5oFqxrkJU_nKatCaYFKV4lvCSDnLahacWApKUSxl71f6656F-TGwCBpVQhe0aYqE3G8JrSDC7nyxoK_kQ6MvA04v5Tgo1E9SlFTYNiQhrSsVKIEIRaYIqJdNAigk9aXTbdxYVGrZJSHfkt0OzOYTi7dlSxJLShpksD-WqB7VHZ0cCKnGCkqXhBeXtHEfto08-5yxBClNWFyOfnrxumMJUkXLprJhQ-P0Ked2FBLSIc1Q-vSHtUkKg9K2lDGaVkkav4ElT6N1qj0MluT4lsF-1sFiYl4HZcwhiCPf579P3v6e5v9-IDtEPrYpQc7RuOGsA2Wa1B5F4LH9t5ZSuQ0WHduyGmw5GawUtm7h5d5X3Q3ScU_qMcfXw</recordid><startdate>20140624</startdate><enddate>20140624</enddate><creator>Logez, Christel</creator><creator>Berger, Sylvie</creator><creator>Legros, Céline</creator><creator>Banères, Jean-Louis</creator><creator>Cohen, William</creator><creator>Delagrange, Philippe</creator><creator>Nosjean, Olivier</creator><creator>Boutin, Jean A</creator><creator>Ferry, Gilles</creator><creator>Simonin, Frédéric</creator><creator>Wagner, Renaud</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>1XC</scope><scope>VOOES</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0001-7078-1285</orcidid></search><sort><creationdate>20140624</creationdate><title>Recombinant human melatonin receptor MT1 isolated in mixed detergents shows pharmacology similar to that in mammalian cell membranes</title><author>Logez, Christel ; Berger, Sylvie ; Legros, Céline ; Banères, Jean-Louis ; Cohen, William ; Delagrange, Philippe ; Nosjean, Olivier ; Boutin, Jean A ; Ferry, Gilles ; Simonin, Frédéric ; Wagner, Renaud</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c726t-7fdd2d5ed6d5df9263288b258e747597453cd9ecd5c72f0ae77fac6c7a2974993</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Animals</topic><topic>Binding</topic><topic>Biology and Life Sciences</topic><topic>Cell Line</topic><topic>Cell Membrane</topic><topic>Cell Membrane - 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Structural and functional information at the molecular level are highly desired for fine characterization of this receptor; however, adequate techniques for isolating soluble MT1 material suitable for biochemical and biophysical studies remain lacking. Here we describe the evaluation of a panel of constructs and host systems for the production of recombinant human MT1 receptors, and the screening of different conditions for their solubilization and purification. Our findings resulted in the establishment of an original strategy using a mixture of Fos14 and CHAPS detergents to extract and purify a recombinant human MT1 from Pichia pastoris membranes. This procedure enabled the recovery of relatively pure, monomeric and ligand-binding active MT1 receptor in the near-milligram range. A comparative study based on extensive ligand-binding characterization highlighted a very close correlation between the pharmacological profiles of MT1 purified from yeast and the same receptor present in mammalian cell membranes. The high quality of the purified MT1 was further confirmed by its ability to activate its cognate Gαi protein partner when reconstituted in lipid discs, thus opening novel paths to investigate this receptor by biochemical and biophysical approaches.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>24959712</pmid><doi>10.1371/journal.pone.0100616</doi><orcidid>https://orcid.org/0000-0001-7078-1285</orcidid><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2014-06, Vol.9 (6), p.e100616-e100616 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1539751854 |
| source | Public Library of Science (PLoS) Journals Open Access; MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Animals Binding Biology and Life Sciences Cell Line Cell Membrane Cell Membrane - chemistry Cell Membrane - metabolism Cell membranes Chemical Sciences CHO Cells Cholesterol Chromatography Circadian rhythms Comparative studies Cricetulus Detergents Detergents - chemistry Escherichia coli Escherichia coli - genetics Escherichia coli - metabolism G protein-coupled receptors G proteins Gene Expression Host systems Humans Ligands Lipids Mammals Melatonin Membranes Mental depression Pharmacology Pichia pastoris Protein Binding Proteins Receptor, Melatonin, MT1 Receptor, Melatonin, MT1 - chemistry Receptor, Melatonin, MT1 - genetics Receptor, Melatonin, MT1 - metabolism Receptors Recombinant Proteins Recombinant Proteins - genetics Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism Sleep Sleep disorders Solubilization Structure-function relationships Yeast Yeasts Yeasts - genetics Yeasts - metabolism |
| title | Recombinant human melatonin receptor MT1 isolated in mixed detergents shows pharmacology similar to that in mammalian cell membranes |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-14T05%3A27%3A49IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Recombinant%20human%20melatonin%20receptor%20MT1%20isolated%20in%20mixed%20detergents%20shows%20pharmacology%20similar%20to%20that%20in%20mammalian%20cell%20membranes&rft.jtitle=PloS%20one&rft.au=Logez,%20Christel&rft.date=2014-06-24&rft.volume=9&rft.issue=6&rft.spage=e100616&rft.epage=e100616&rft.pages=e100616-e100616&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0100616&rft_dat=%3Cgale_plos_%3EA418127143%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1539751854&rft_id=info:pmid/24959712&rft_galeid=A418127143&rft_doaj_id=oai_doaj_org_article_961a2e8080f24c94a99bea2e9fb8eaad&rfr_iscdi=true |