Baculovirus-mediated expression of retinoic acid receptor type gamma in cultured insect cells reveals a difference in specific DNA-binding behavior with the 1,25-dihydroxyvitamin D3 receptor
The baculovirus genetic expression system has been used to produce murine retinoic acid receptor (RAR) type gamma in Spodoptera frugiperda insect cells and Manduca sexta insect larvae. A hydroxyapatite binding assay revealed production levels of 300 pmol of unoccupied receptor per mg of protein in i...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 1992-11, Vol.89 (21), p.10282-10286 |
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| creator | ROSS, T. K PRAHL, J. M HERZBERG, I. M DELUCA, H. F |
| description | The baculovirus genetic expression system has been used to produce murine retinoic acid receptor (RAR) type gamma in Spodoptera frugiperda insect cells and Manduca sexta insect larvae. A hydroxyapatite binding assay revealed production levels of 300 pmol of unoccupied receptor per mg of protein in insect cells, whereas levels from infected insect larvae were found to average 100 pmol of RAR gamma per mg of protein. The cytosolic preparation from infected insect cells exhibited an equilibrium dissociation constant of 2.1 nM as determined by a retinoic acid saturation analysis plotted by the method of Scatchard. A polyclonal antibody directed against RAR gamma recognized the recombinant receptor protein as a 54,000-Da species. Electrophoretic mobility shift analyses demonstrated that protein extracts from RAR gamma-producing insect cells or larvae are capable of retinoic acid responsive element binding. This contrasts with the specific DNA-binding behavior of the insect cell-produced vitamin D receptor, which requires the presence of a mammalian-derived nuclear accessory protein. This distinction between RAR gamma and the vitamin D receptor suggests a difference in the molecular requirements by these two receptors for specific binding of their respective DNA response elements. |
| doi_str_mv | 10.1073/pnas.89.21.10282 |
| format | Article |
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K ; PRAHL, J. M ; HERZBERG, I. M ; DELUCA, H. F</creator><creatorcontrib>ROSS, T. K ; PRAHL, J. M ; HERZBERG, I. M ; DELUCA, H. F ; University of Wisconsin, Madison, WI ; Northeastern Forest Experiment Station, Radnor, Pa. (USA)</creatorcontrib><description>The baculovirus genetic expression system has been used to produce murine retinoic acid receptor (RAR) type gamma in Spodoptera frugiperda insect cells and Manduca sexta insect larvae. A hydroxyapatite binding assay revealed production levels of 300 pmol of unoccupied receptor per mg of protein in insect cells, whereas levels from infected insect larvae were found to average 100 pmol of RAR gamma per mg of protein. The cytosolic preparation from infected insect cells exhibited an equilibrium dissociation constant of 2.1 nM as determined by a retinoic acid saturation analysis plotted by the method of Scatchard. A polyclonal antibody directed against RAR gamma recognized the recombinant receptor protein as a 54,000-Da species. Electrophoretic mobility shift analyses demonstrated that protein extracts from RAR gamma-producing insect cells or larvae are capable of retinoic acid responsive element binding. This contrasts with the specific DNA-binding behavior of the insect cell-produced vitamin D receptor, which requires the presence of a mammalian-derived nuclear accessory protein. This distinction between RAR gamma and the vitamin D receptor suggests a difference in the molecular requirements by these two receptors for specific binding of their respective DNA response elements.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.89.21.10282</identifier><identifier>PMID: 1332041</identifier><identifier>CODEN: PNASA6</identifier><language>eng</language><publisher>Washington, DC: National Acad Sciences</publisher><subject>adn ; Animals ; autographa californica ; baculoviridae ; Baculoviridae - genetics ; Biological and medical sciences ; Blotting, Western ; Calcitriol - metabolism ; Carrier Proteins - classification ; Carrier Proteins - genetics ; Carrier Proteins - metabolism ; Cell Line ; Cell receptors ; Cell structures and functions ; Chromatography, Affinity ; dna ; DNA-Binding Proteins - genetics ; DNA-Binding Proteins - metabolism ; Electrophoresis, Polyacrylamide Gel ; expresion genica ; expression des genes ; Fundamental and applied biological sciences. Psychology ; gene expression ; Kinetics ; manduca sexta ; Mice ; Miscellaneous ; Molecular and cellular biology ; Molecular Weight ; Moths ; Receptors, Calcitriol ; Receptors, Retinoic Acid ; Receptors, Steroid - metabolism ; Restriction Mapping ; retinol ; spodoptera frugiperda ; Transfection ; Tretinoin - metabolism</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 1992-11, Vol.89 (21), p.10282-10286</ispartof><rights>1993 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c430t-2ca218e6407a5c4b81297103d74aee923ccf487d5318fac04b2f70c423e2c8b33</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/89/21.cover.gif</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC50322/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC50322/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=4476323$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/1332041$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>ROSS, T. K</creatorcontrib><creatorcontrib>PRAHL, J. M</creatorcontrib><creatorcontrib>HERZBERG, I. M</creatorcontrib><creatorcontrib>DELUCA, H. F</creatorcontrib><creatorcontrib>University of Wisconsin, Madison, WI</creatorcontrib><creatorcontrib>Northeastern Forest Experiment Station, Radnor, Pa. (USA)</creatorcontrib><title>Baculovirus-mediated expression of retinoic acid receptor type gamma in cultured insect cells reveals a difference in specific DNA-binding behavior with the 1,25-dihydroxyvitamin D3 receptor</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>The baculovirus genetic expression system has been used to produce murine retinoic acid receptor (RAR) type gamma in Spodoptera frugiperda insect cells and Manduca sexta insect larvae. A hydroxyapatite binding assay revealed production levels of 300 pmol of unoccupied receptor per mg of protein in insect cells, whereas levels from infected insect larvae were found to average 100 pmol of RAR gamma per mg of protein. The cytosolic preparation from infected insect cells exhibited an equilibrium dissociation constant of 2.1 nM as determined by a retinoic acid saturation analysis plotted by the method of Scatchard. A polyclonal antibody directed against RAR gamma recognized the recombinant receptor protein as a 54,000-Da species. Electrophoretic mobility shift analyses demonstrated that protein extracts from RAR gamma-producing insect cells or larvae are capable of retinoic acid responsive element binding. This contrasts with the specific DNA-binding behavior of the insect cell-produced vitamin D receptor, which requires the presence of a mammalian-derived nuclear accessory protein. This distinction between RAR gamma and the vitamin D receptor suggests a difference in the molecular requirements by these two receptors for specific binding of their respective DNA response elements.</description><subject>adn</subject><subject>Animals</subject><subject>autographa californica</subject><subject>baculoviridae</subject><subject>Baculoviridae - genetics</subject><subject>Biological and medical sciences</subject><subject>Blotting, Western</subject><subject>Calcitriol - metabolism</subject><subject>Carrier Proteins - classification</subject><subject>Carrier Proteins - genetics</subject><subject>Carrier Proteins - metabolism</subject><subject>Cell Line</subject><subject>Cell receptors</subject><subject>Cell structures and functions</subject><subject>Chromatography, Affinity</subject><subject>dna</subject><subject>DNA-Binding Proteins - genetics</subject><subject>DNA-Binding Proteins - metabolism</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>expresion genica</subject><subject>expression des genes</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>gene expression</subject><subject>Kinetics</subject><subject>manduca sexta</subject><subject>Mice</subject><subject>Miscellaneous</subject><subject>Molecular and cellular biology</subject><subject>Molecular Weight</subject><subject>Moths</subject><subject>Receptors, Calcitriol</subject><subject>Receptors, Retinoic Acid</subject><subject>Receptors, Steroid - metabolism</subject><subject>Restriction Mapping</subject><subject>retinol</subject><subject>spodoptera frugiperda</subject><subject>Transfection</subject><subject>Tretinoin - metabolism</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1992</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kUtv1DAUhSMEKqWwZwN4gRALMviViSOxKS0vqYIFdG05zvWMUWKntjPM_Dl-Gw4zDGXDyrLud8659imKxwQvCK7Z69GpuBDNgpJ8p4LeKU4Jbki55A2-W5xiTOtScMrvFw9i_I4xbiqBT4oTwhjFnJwWP98qPfV-Y8MUywE6qxJ0CLZjgBitd8gbFCBZ561GStsu3zSMyQeUdiOglRoGhaxD2SVNIWuti6AT0tD3McMbUPlUqLPGQACnYabjCNqabHn5-bxsreusW6EW1mpjs_MPm9YorQGRV7QqO7vedcFvdxub1JDFl-y4xMPinsn-8OhwnhXX7999u_hYXn358Oni_KrUnOFUUq0oEbDkuFaV5q0gtKkJZl3NFUBDmdaGi7qrGBFGacxbamqsOWVAtWgZOyve7H3Hqc2_pMGloHo5BjuosJNeWfnvxNm1XPmNrDCjNMtfHOTB30wQkxxsnH9IOfBTlHWuQ2BSZRDvQR18jAHMMYJgOTcu58alaCQl8nfjWfLk9mp_BfuK8_z5Ya6iVr0Jymkbjxjn9ZLR-YUvD9gc8Gd6K0iaqe8TbFNGn_0fzcTTPWGUl2oVct71V9I0DONlVefAXyTR2p8</recordid><startdate>19921101</startdate><enddate>19921101</enddate><creator>ROSS, T. K</creator><creator>PRAHL, J. M</creator><creator>HERZBERG, I. M</creator><creator>DELUCA, H. F</creator><general>National Acad Sciences</general><general>National Academy of Sciences of the United States of America</general><scope>FBQ</scope><scope>IQODW</scope><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>7X8</scope><scope>5PM</scope></search><sort><creationdate>19921101</creationdate><title>Baculovirus-mediated expression of retinoic acid receptor type gamma in cultured insect cells reveals a difference in specific DNA-binding behavior with the 1,25-dihydroxyvitamin D3 receptor</title><author>ROSS, T. K ; PRAHL, J. M ; HERZBERG, I. M ; DELUCA, H. F</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c430t-2ca218e6407a5c4b81297103d74aee923ccf487d5318fac04b2f70c423e2c8b33</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1992</creationdate><topic>adn</topic><topic>Animals</topic><topic>autographa californica</topic><topic>baculoviridae</topic><topic>Baculoviridae - genetics</topic><topic>Biological and medical sciences</topic><topic>Blotting, Western</topic><topic>Calcitriol - metabolism</topic><topic>Carrier Proteins - classification</topic><topic>Carrier Proteins - genetics</topic><topic>Carrier Proteins - metabolism</topic><topic>Cell Line</topic><topic>Cell receptors</topic><topic>Cell structures and functions</topic><topic>Chromatography, Affinity</topic><topic>dna</topic><topic>DNA-Binding Proteins - genetics</topic><topic>DNA-Binding Proteins - metabolism</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>expresion genica</topic><topic>expression des genes</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>gene expression</topic><topic>Kinetics</topic><topic>manduca sexta</topic><topic>Mice</topic><topic>Miscellaneous</topic><topic>Molecular and cellular biology</topic><topic>Molecular Weight</topic><topic>Moths</topic><topic>Receptors, Calcitriol</topic><topic>Receptors, Retinoic Acid</topic><topic>Receptors, Steroid - metabolism</topic><topic>Restriction Mapping</topic><topic>retinol</topic><topic>spodoptera frugiperda</topic><topic>Transfection</topic><topic>Tretinoin - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>ROSS, T. K</creatorcontrib><creatorcontrib>PRAHL, J. M</creatorcontrib><creatorcontrib>HERZBERG, I. M</creatorcontrib><creatorcontrib>DELUCA, H. F</creatorcontrib><creatorcontrib>University of Wisconsin, Madison, WI</creatorcontrib><creatorcontrib>Northeastern Forest Experiment Station, Radnor, Pa. (USA)</creatorcontrib><collection>AGRIS</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>ROSS, T. K</au><au>PRAHL, J. M</au><au>HERZBERG, I. M</au><au>DELUCA, H. F</au><aucorp>University of Wisconsin, Madison, WI</aucorp><aucorp>Northeastern Forest Experiment Station, Radnor, Pa. (USA)</aucorp><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Baculovirus-mediated expression of retinoic acid receptor type gamma in cultured insect cells reveals a difference in specific DNA-binding behavior with the 1,25-dihydroxyvitamin D3 receptor</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>1992-11-01</date><risdate>1992</risdate><volume>89</volume><issue>21</issue><spage>10282</spage><epage>10286</epage><pages>10282-10286</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><coden>PNASA6</coden><abstract>The baculovirus genetic expression system has been used to produce murine retinoic acid receptor (RAR) type gamma in Spodoptera frugiperda insect cells and Manduca sexta insect larvae. A hydroxyapatite binding assay revealed production levels of 300 pmol of unoccupied receptor per mg of protein in insect cells, whereas levels from infected insect larvae were found to average 100 pmol of RAR gamma per mg of protein. The cytosolic preparation from infected insect cells exhibited an equilibrium dissociation constant of 2.1 nM as determined by a retinoic acid saturation analysis plotted by the method of Scatchard. A polyclonal antibody directed against RAR gamma recognized the recombinant receptor protein as a 54,000-Da species. Electrophoretic mobility shift analyses demonstrated that protein extracts from RAR gamma-producing insect cells or larvae are capable of retinoic acid responsive element binding. This contrasts with the specific DNA-binding behavior of the insect cell-produced vitamin D receptor, which requires the presence of a mammalian-derived nuclear accessory protein. This distinction between RAR gamma and the vitamin D receptor suggests a difference in the molecular requirements by these two receptors for specific binding of their respective DNA response elements.</abstract><cop>Washington, DC</cop><pub>National Acad Sciences</pub><pmid>1332041</pmid><doi>10.1073/pnas.89.21.10282</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| source | Jstor Complete Legacy; MEDLINE; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry |
| subjects | adn Animals autographa californica baculoviridae Baculoviridae - genetics Biological and medical sciences Blotting, Western Calcitriol - metabolism Carrier Proteins - classification Carrier Proteins - genetics Carrier Proteins - metabolism Cell Line Cell receptors Cell structures and functions Chromatography, Affinity dna DNA-Binding Proteins - genetics DNA-Binding Proteins - metabolism Electrophoresis, Polyacrylamide Gel expresion genica expression des genes Fundamental and applied biological sciences. Psychology gene expression Kinetics manduca sexta Mice Miscellaneous Molecular and cellular biology Molecular Weight Moths Receptors, Calcitriol Receptors, Retinoic Acid Receptors, Steroid - metabolism Restriction Mapping retinol spodoptera frugiperda Transfection Tretinoin - metabolism |
| title | Baculovirus-mediated expression of retinoic acid receptor type gamma in cultured insect cells reveals a difference in specific DNA-binding behavior with the 1,25-dihydroxyvitamin D3 receptor |
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