Characterization of the recombinant human receptor for Escherichia coli heat-stable enterotoxin
We report here the molecular characterization of a recombinant cell line (293-STaR) expressing the heat-stable enterotoxin receptor (STaR) from human intestine. We have compared the 293-STaR cell line with the human colonic cell line T84 that endogenously expresses STa binding sites. Scatchard analy...
Gespeichert in:
| Veröffentlicht in: | The Journal of biological chemistry 1992-04, Vol.267 (10), p.6479-6482 |
|---|---|
| Hauptverfasser: | , , , , , |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | 6482 |
|---|---|
| container_issue | 10 |
| container_start_page | 6479 |
| container_title | The Journal of biological chemistry |
| container_volume | 267 |
| creator | de Sauvage, F J Horuk, R Bennett, G Quan, C Burnier, J P Goeddel, D V |
| description | We report here the molecular characterization of a recombinant cell line (293-STaR) expressing the heat-stable enterotoxin
receptor (STaR) from human intestine. We have compared the 293-STaR cell line with the human colonic cell line T84 that endogenously
expresses STa binding sites. Scatchard analysis of displacement binding studies revealed a single STa binding site with an
affinity (Ki) of 97 pM in 293-STaR compared with 55 pM in T84 cells. Saturation isotherms of STa binding gave a Kd of 94 pM
for the cloned receptor expressed in 293 cells and 166 pM for the receptor present in T84 cells. Kinetic measurements of STa
binding to 293-STaR gave an association rate constant, K1, of 2.4 x 10(8) M-1 min-1 and a dissociation rate constant, K2,
of 0.016 min-1. The half-time of dissociation was 43 min, and the Kd calculated from the ratio of the kinetic constants was
67 pM. The pH profile of STa binding showed that the number of STa binding sites is increased 3-fold at pH 4.0 compared with
pH 7.0, with no effect on binding affinity. A polyclonal antibody directed against the extracellular domain of STaR immunoprecipitated
two proteins of approximately 140 and 160 kDa from both 293-STaR and T84 cells. Cross-linking of 125I-STa to 293-STaR cells
resulted in the labeling of proteins with a molecular mass of approximately 153, 133, 81, 68, 56, and 49 kDa, the two smallest
being the more abundant. Similar results have been reported for the STaR present on rat brush border membranes. These data
suggest that the STaR-guanylyl cyclase identified by molecular cloning is the only receptor for STa present in T84 cells. |
| doi_str_mv | 10.1016/S0021-9258(19)50452-5 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_72868052</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>72868052</sourcerecordid><originalsourceid>FETCH-LOGICAL-c410t-cac3802a64cf6dde0fc16f10c38ad320f09b67d7232ab6cc1934a3840848fa373</originalsourceid><addsrcrecordid>eNqFUdGK1TAQDeKyXlc_YaEgyPpQnUmaNH2Uy64KCz6o4FuYpomNtM01yUXdr7fdu-ijA8PAzDln4BzGLhFeI6B68wmAY91xqa-weyWhkbyWj9gOQYtaSPz6mO3-Qp6wpzl_h7WaDs_ZOQoUAHLHzH6kRLa4FO6ohLhU0VdldFVyNs59WGgp1Xicadk27lBiqvza19mOK8eOgSobp1CNjkqdC_WTq9yy6sUSf4XlGTvzNGX3_GFesC8315_37-vbj-8-7N_e1rZBKLUlKzRwUo31ahgceIvKI6xbGgQHD12v2qHlglOvrMVONCR0A7rRnkQrLtjLk-4hxR9Hl4uZQ7Zummhx8ZhNy7XSIPl_gaiEarXeFOUJaFPMOTlvDinMlH4bBLMlYO4TMJu9Bjtzn4CRK-_y4cGxn93wj3WyfL2_ON3H8G38GZIzfYirmbPhqt20VdN24g_BTI4c</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>16367887</pqid></control><display><type>article</type><title>Characterization of the recombinant human receptor for Escherichia coli heat-stable enterotoxin</title><source>MEDLINE</source><source>Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals</source><source>Alma/SFX Local Collection</source><creator>de Sauvage, F J ; Horuk, R ; Bennett, G ; Quan, C ; Burnier, J P ; Goeddel, D V</creator><creatorcontrib>de Sauvage, F J ; Horuk, R ; Bennett, G ; Quan, C ; Burnier, J P ; Goeddel, D V</creatorcontrib><description>We report here the molecular characterization of a recombinant cell line (293-STaR) expressing the heat-stable enterotoxin
receptor (STaR) from human intestine. We have compared the 293-STaR cell line with the human colonic cell line T84 that endogenously
expresses STa binding sites. Scatchard analysis of displacement binding studies revealed a single STa binding site with an
affinity (Ki) of 97 pM in 293-STaR compared with 55 pM in T84 cells. Saturation isotherms of STa binding gave a Kd of 94 pM
for the cloned receptor expressed in 293 cells and 166 pM for the receptor present in T84 cells. Kinetic measurements of STa
binding to 293-STaR gave an association rate constant, K1, of 2.4 x 10(8) M-1 min-1 and a dissociation rate constant, K2,
of 0.016 min-1. The half-time of dissociation was 43 min, and the Kd calculated from the ratio of the kinetic constants was
67 pM. The pH profile of STa binding showed that the number of STa binding sites is increased 3-fold at pH 4.0 compared with
pH 7.0, with no effect on binding affinity. A polyclonal antibody directed against the extracellular domain of STaR immunoprecipitated
two proteins of approximately 140 and 160 kDa from both 293-STaR and T84 cells. Cross-linking of 125I-STa to 293-STaR cells
resulted in the labeling of proteins with a molecular mass of approximately 153, 133, 81, 68, 56, and 49 kDa, the two smallest
being the more abundant. Similar results have been reported for the STaR present on rat brush border membranes. These data
suggest that the STaR-guanylyl cyclase identified by molecular cloning is the only receptor for STa present in T84 cells.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/S0021-9258(19)50452-5</identifier><identifier>PMID: 1313005</identifier><language>eng</language><publisher>United States: American Society for Biochemistry and Molecular Biology</publisher><subject>Bacterial Toxins - metabolism ; Binding Sites ; biochemical characteristics ; Cell Line ; Colon - metabolism ; Electrophoresis, Polyacrylamide Gel ; enterotoxins ; Enterotoxins - metabolism ; Escherichia coli ; Escherichia coli - metabolism ; Escherichia coli Proteins ; Gene Expression ; Guanylate Cyclase ; Humans ; Hydrogen-Ion Concentration ; Kinetics ; man ; membrane proteins ; Precipitin Tests ; receptors ; Receptors, Cell Surface - genetics ; Receptors, Cell Surface - metabolism ; Receptors, Enterotoxin ; Receptors, Guanylate Cyclase-Coupled ; Receptors, Peptide ; recombinant ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; T84 cells</subject><ispartof>The Journal of biological chemistry, 1992-04, Vol.267 (10), p.6479-6482</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c410t-cac3802a64cf6dde0fc16f10c38ad320f09b67d7232ab6cc1934a3840848fa373</citedby><cites>FETCH-LOGICAL-c410t-cac3802a64cf6dde0fc16f10c38ad320f09b67d7232ab6cc1934a3840848fa373</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/1313005$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>de Sauvage, F J</creatorcontrib><creatorcontrib>Horuk, R</creatorcontrib><creatorcontrib>Bennett, G</creatorcontrib><creatorcontrib>Quan, C</creatorcontrib><creatorcontrib>Burnier, J P</creatorcontrib><creatorcontrib>Goeddel, D V</creatorcontrib><title>Characterization of the recombinant human receptor for Escherichia coli heat-stable enterotoxin</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>We report here the molecular characterization of a recombinant cell line (293-STaR) expressing the heat-stable enterotoxin
receptor (STaR) from human intestine. We have compared the 293-STaR cell line with the human colonic cell line T84 that endogenously
expresses STa binding sites. Scatchard analysis of displacement binding studies revealed a single STa binding site with an
affinity (Ki) of 97 pM in 293-STaR compared with 55 pM in T84 cells. Saturation isotherms of STa binding gave a Kd of 94 pM
for the cloned receptor expressed in 293 cells and 166 pM for the receptor present in T84 cells. Kinetic measurements of STa
binding to 293-STaR gave an association rate constant, K1, of 2.4 x 10(8) M-1 min-1 and a dissociation rate constant, K2,
of 0.016 min-1. The half-time of dissociation was 43 min, and the Kd calculated from the ratio of the kinetic constants was
67 pM. The pH profile of STa binding showed that the number of STa binding sites is increased 3-fold at pH 4.0 compared with
pH 7.0, with no effect on binding affinity. A polyclonal antibody directed against the extracellular domain of STaR immunoprecipitated
two proteins of approximately 140 and 160 kDa from both 293-STaR and T84 cells. Cross-linking of 125I-STa to 293-STaR cells
resulted in the labeling of proteins with a molecular mass of approximately 153, 133, 81, 68, 56, and 49 kDa, the two smallest
being the more abundant. Similar results have been reported for the STaR present on rat brush border membranes. These data
suggest that the STaR-guanylyl cyclase identified by molecular cloning is the only receptor for STa present in T84 cells.</description><subject>Bacterial Toxins - metabolism</subject><subject>Binding Sites</subject><subject>biochemical characteristics</subject><subject>Cell Line</subject><subject>Colon - metabolism</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>enterotoxins</subject><subject>Enterotoxins - metabolism</subject><subject>Escherichia coli</subject><subject>Escherichia coli - metabolism</subject><subject>Escherichia coli Proteins</subject><subject>Gene Expression</subject><subject>Guanylate Cyclase</subject><subject>Humans</subject><subject>Hydrogen-Ion Concentration</subject><subject>Kinetics</subject><subject>man</subject><subject>membrane proteins</subject><subject>Precipitin Tests</subject><subject>receptors</subject><subject>Receptors, Cell Surface - genetics</subject><subject>Receptors, Cell Surface - metabolism</subject><subject>Receptors, Enterotoxin</subject><subject>Receptors, Guanylate Cyclase-Coupled</subject><subject>Receptors, Peptide</subject><subject>recombinant</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>T84 cells</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1992</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFUdGK1TAQDeKyXlc_YaEgyPpQnUmaNH2Uy64KCz6o4FuYpomNtM01yUXdr7fdu-ijA8PAzDln4BzGLhFeI6B68wmAY91xqa-weyWhkbyWj9gOQYtaSPz6mO3-Qp6wpzl_h7WaDs_ZOQoUAHLHzH6kRLa4FO6ohLhU0VdldFVyNs59WGgp1Xicadk27lBiqvza19mOK8eOgSobp1CNjkqdC_WTq9yy6sUSf4XlGTvzNGX3_GFesC8315_37-vbj-8-7N_e1rZBKLUlKzRwUo31ahgceIvKI6xbGgQHD12v2qHlglOvrMVONCR0A7rRnkQrLtjLk-4hxR9Hl4uZQ7Zummhx8ZhNy7XSIPl_gaiEarXeFOUJaFPMOTlvDinMlH4bBLMlYO4TMJu9Bjtzn4CRK-_y4cGxn93wj3WyfL2_ON3H8G38GZIzfYirmbPhqt20VdN24g_BTI4c</recordid><startdate>19920405</startdate><enddate>19920405</enddate><creator>de Sauvage, F J</creator><creator>Horuk, R</creator><creator>Bennett, G</creator><creator>Quan, C</creator><creator>Burnier, J P</creator><creator>Goeddel, D V</creator><general>American Society for Biochemistry and Molecular Biology</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>8FD</scope><scope>FR3</scope><scope>M7Z</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>19920405</creationdate><title>Characterization of the recombinant human receptor for Escherichia coli heat-stable enterotoxin</title><author>de Sauvage, F J ; Horuk, R ; Bennett, G ; Quan, C ; Burnier, J P ; Goeddel, D V</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c410t-cac3802a64cf6dde0fc16f10c38ad320f09b67d7232ab6cc1934a3840848fa373</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1992</creationdate><topic>Bacterial Toxins - metabolism</topic><topic>Binding Sites</topic><topic>biochemical characteristics</topic><topic>Cell Line</topic><topic>Colon - metabolism</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>enterotoxins</topic><topic>Enterotoxins - metabolism</topic><topic>Escherichia coli</topic><topic>Escherichia coli - metabolism</topic><topic>Escherichia coli Proteins</topic><topic>Gene Expression</topic><topic>Guanylate Cyclase</topic><topic>Humans</topic><topic>Hydrogen-Ion Concentration</topic><topic>Kinetics</topic><topic>man</topic><topic>membrane proteins</topic><topic>Precipitin Tests</topic><topic>receptors</topic><topic>Receptors, Cell Surface - genetics</topic><topic>Receptors, Cell Surface - metabolism</topic><topic>Receptors, Enterotoxin</topic><topic>Receptors, Guanylate Cyclase-Coupled</topic><topic>Receptors, Peptide</topic><topic>recombinant</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>T84 cells</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>de Sauvage, F J</creatorcontrib><creatorcontrib>Horuk, R</creatorcontrib><creatorcontrib>Bennett, G</creatorcontrib><creatorcontrib>Quan, C</creatorcontrib><creatorcontrib>Burnier, J P</creatorcontrib><creatorcontrib>Goeddel, D V</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biochemistry Abstracts 1</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>de Sauvage, F J</au><au>Horuk, R</au><au>Bennett, G</au><au>Quan, C</au><au>Burnier, J P</au><au>Goeddel, D V</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Characterization of the recombinant human receptor for Escherichia coli heat-stable enterotoxin</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1992-04-05</date><risdate>1992</risdate><volume>267</volume><issue>10</issue><spage>6479</spage><epage>6482</epage><pages>6479-6482</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>We report here the molecular characterization of a recombinant cell line (293-STaR) expressing the heat-stable enterotoxin
receptor (STaR) from human intestine. We have compared the 293-STaR cell line with the human colonic cell line T84 that endogenously
expresses STa binding sites. Scatchard analysis of displacement binding studies revealed a single STa binding site with an
affinity (Ki) of 97 pM in 293-STaR compared with 55 pM in T84 cells. Saturation isotherms of STa binding gave a Kd of 94 pM
for the cloned receptor expressed in 293 cells and 166 pM for the receptor present in T84 cells. Kinetic measurements of STa
binding to 293-STaR gave an association rate constant, K1, of 2.4 x 10(8) M-1 min-1 and a dissociation rate constant, K2,
of 0.016 min-1. The half-time of dissociation was 43 min, and the Kd calculated from the ratio of the kinetic constants was
67 pM. The pH profile of STa binding showed that the number of STa binding sites is increased 3-fold at pH 4.0 compared with
pH 7.0, with no effect on binding affinity. A polyclonal antibody directed against the extracellular domain of STaR immunoprecipitated
two proteins of approximately 140 and 160 kDa from both 293-STaR and T84 cells. Cross-linking of 125I-STa to 293-STaR cells
resulted in the labeling of proteins with a molecular mass of approximately 153, 133, 81, 68, 56, and 49 kDa, the two smallest
being the more abundant. Similar results have been reported for the STaR present on rat brush border membranes. These data
suggest that the STaR-guanylyl cyclase identified by molecular cloning is the only receptor for STa present in T84 cells.</abstract><cop>United States</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>1313005</pmid><doi>10.1016/S0021-9258(19)50452-5</doi><tpages>4</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0021-9258 |
| ispartof | The Journal of biological chemistry, 1992-04, Vol.267 (10), p.6479-6482 |
| issn | 0021-9258 1083-351X |
| language | eng |
| recordid | cdi_proquest_miscellaneous_72868052 |
| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | Bacterial Toxins - metabolism Binding Sites biochemical characteristics Cell Line Colon - metabolism Electrophoresis, Polyacrylamide Gel enterotoxins Enterotoxins - metabolism Escherichia coli Escherichia coli - metabolism Escherichia coli Proteins Gene Expression Guanylate Cyclase Humans Hydrogen-Ion Concentration Kinetics man membrane proteins Precipitin Tests receptors Receptors, Cell Surface - genetics Receptors, Cell Surface - metabolism Receptors, Enterotoxin Receptors, Guanylate Cyclase-Coupled Receptors, Peptide recombinant Recombinant Proteins - genetics Recombinant Proteins - metabolism T84 cells |
| title | Characterization of the recombinant human receptor for Escherichia coli heat-stable enterotoxin |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-04T20%3A10%3A48IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Characterization%20of%20the%20recombinant%20human%20receptor%20for%20Escherichia%20coli%20heat-stable%20enterotoxin&rft.jtitle=The%20Journal%20of%20biological%20chemistry&rft.au=de%20Sauvage,%20F%20J&rft.date=1992-04-05&rft.volume=267&rft.issue=10&rft.spage=6479&rft.epage=6482&rft.pages=6479-6482&rft.issn=0021-9258&rft.eissn=1083-351X&rft_id=info:doi/10.1016/S0021-9258(19)50452-5&rft_dat=%3Cproquest_cross%3E72868052%3C/proquest_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=16367887&rft_id=info:pmid/1313005&rfr_iscdi=true |