Characterization of a recombinant extracellular domain of the type 1 tumor necrosis factor receptor: evidence for tumor necrosis factor .alpha. induced receptor aggregation
An expression plasmid encoding the extracellular portion of the human tumor necrosis factor (TNF) type 1 receptor (TNF-R1) was constructed and used to generate a stable cell line secreting soluble TNF-R1 (sTNF-R1). The sTNF-R1 was purified, and its biochemical properties and its interactions with hu...
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| Veröffentlicht in: | Biochemistry (Easton) 1992-02, Vol.31 (4), p.1134-1141 |
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| container_title | Biochemistry (Easton) |
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| creator | Pennica, Diane Kohr, William J Fendly, Brian M Shire, Steven J Raab, Helga E Borchardt, Paul E Lewis, Martyn Goeddel, David V |
| description | An expression plasmid encoding the extracellular portion of the human tumor necrosis factor (TNF) type 1 receptor (TNF-R1) was constructed and used to generate a stable cell line secreting soluble TNF-R1 (sTNF-R1). The sTNF-R1 was purified, and its biochemical properties and its interactions with human TNF-alpha were examined. SDS-PAGE resolved the purified sTNF-R1 into three bands of approximate Mr 24,200, 28,200, and 32,800. Sedimentation equilibrium analysis gave a molecular weight of 25,000 for sTNF-R1 whereas the molecular weight obtained by gel filtration chromatography was approximately 55,000-60,000. Scatchard analysis of [125I]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (Kd = 93 pM), comparable to that observed for the intact receptor on whole cells. Competitive binding experiments showed that sTNF-R1 has a 50-60-fold higher affinity for TNF-alpha than for TNF-beta, in contrast to the equal affinities of TNF-alpha and TNF-beta for the full-length TNF-R1 transiently expressed in mammalian cells. The sTNF-R1 was found to block the cytotoxicity of TNF-alpha and TNF-beta on a murine L-M cell assay. The sizes of the sTNF-R1.TNF-alpha complex determined by gel filtration chromatography and sedimentation equilibrium were approximately 141 and 115 kDa, respectively. The stoichiometry of the complex was examined by Scatchard analysis, size-exclusion chromatography, HPLC separation, amino acid composition, sequence analysis, and sedimentation equilibrium. The data from these studies suggest that at least two molecules of sTNF-R1 can bind to a single TNF-alpha trimer. |
| doi_str_mv | 10.1021/bi00119a023 |
| format | Article |
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The sTNF-R1 was purified, and its biochemical properties and its interactions with human TNF-alpha were examined. SDS-PAGE resolved the purified sTNF-R1 into three bands of approximate Mr 24,200, 28,200, and 32,800. Sedimentation equilibrium analysis gave a molecular weight of 25,000 for sTNF-R1 whereas the molecular weight obtained by gel filtration chromatography was approximately 55,000-60,000. Scatchard analysis of [125I]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (Kd = 93 pM), comparable to that observed for the intact receptor on whole cells. Competitive binding experiments showed that sTNF-R1 has a 50-60-fold higher affinity for TNF-alpha than for TNF-beta, in contrast to the equal affinities of TNF-alpha and TNF-beta for the full-length TNF-R1 transiently expressed in mammalian cells. The sTNF-R1 was found to block the cytotoxicity of TNF-alpha and TNF-beta on a murine L-M cell assay. The sizes of the sTNF-R1.TNF-alpha complex determined by gel filtration chromatography and sedimentation equilibrium were approximately 141 and 115 kDa, respectively. The stoichiometry of the complex was examined by Scatchard analysis, size-exclusion chromatography, HPLC separation, amino acid composition, sequence analysis, and sedimentation equilibrium. The data from these studies suggest that at least two molecules of sTNF-R1 can bind to a single TNF-alpha trimer.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi00119a023</identifier><identifier>PMID: 1310420</identifier><language>eng</language><publisher>Washington, DC: American Chemical Society</publisher><subject>550201 - Biochemistry- Tracer Techniques ; Amino Acid Sequence ; Animals ; BASIC BIOLOGICAL SCIENCES ; BETA DECAY RADIOISOTOPES ; BIOCHEMICAL REACTION KINETICS ; Biological and medical sciences ; CELL CONSTITUENTS ; Cell Line ; Cell receptors ; Cell structures and functions ; Chromatography, Gel ; Cytotoxicity Tests, Immunologic ; DAYS LIVING RADIOISOTOPES ; DISEASES ; ELECTRON CAPTURE RADIOISOTOPES ; ELECTROPHORESIS ; Fundamental and applied biological sciences. Psychology ; GROWTH FACTORS ; Hormone receptors. Growth factor receptors. Cytokine receptors. Prostaglandin receptors ; Humans ; INTERMEDIATE MASS NUCLEI ; INTERNAL CONVERSION RADIOISOTOPES ; IODINE 125 ; IODINE ISOTOPES ; ISOTOPE APPLICATIONS ; ISOTOPES ; KINETICS ; LYMPHOKINES ; Mice ; MITOGENS ; Molecular and cellular biology ; Molecular Weight ; NECROSIS ; NEOPLASMS ; NUCLEI ; ODD-EVEN NUCLEI ; ORGANIC COMPOUNDS ; PATHOLOGICAL CHANGES ; PLASMIDS ; PROTEINS ; RADIOISOTOPES ; RADIORECEPTOR ASSAY ; REACTION KINETICS ; Receptor Aggregation ; Receptors, Cell Surface - chemistry ; Receptors, Cell Surface - drug effects ; Receptors, Cell Surface - isolation & purification ; Receptors, Tumor Necrosis Factor ; Recombinant Proteins - chemistry ; Recombinant Proteins - drug effects ; Recombinant Proteins - isolation & purification ; signal transduction ; Solubility ; TRACER TECHNIQUES ; tumor necrosis factor ; Tumor Necrosis Factor-alpha - pharmacology ; Ultracentrifugation</subject><ispartof>Biochemistry (Easton), 1992-02, Vol.31 (4), p.1134-1141</ispartof><rights>1992 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a441t-2d7e5a5d3a8bbb8b23364bb5d6baf0f7407347819f022b65df2b2d31c079c9ad3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/bi00119a023$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/bi00119a023$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>230,314,780,784,885,2765,27076,27924,27925,56738,56788</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=5264188$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/1310420$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://www.osti.gov/biblio/5488773$$D View this record in Osti.gov$$Hfree_for_read</backlink></links><search><creatorcontrib>Pennica, Diane</creatorcontrib><creatorcontrib>Kohr, William J</creatorcontrib><creatorcontrib>Fendly, Brian M</creatorcontrib><creatorcontrib>Shire, Steven J</creatorcontrib><creatorcontrib>Raab, Helga E</creatorcontrib><creatorcontrib>Borchardt, Paul E</creatorcontrib><creatorcontrib>Lewis, Martyn</creatorcontrib><creatorcontrib>Goeddel, David V</creatorcontrib><title>Characterization of a recombinant extracellular domain of the type 1 tumor necrosis factor receptor: evidence for tumor necrosis factor .alpha. induced receptor aggregation</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>An expression plasmid encoding the extracellular portion of the human tumor necrosis factor (TNF) type 1 receptor (TNF-R1) was constructed and used to generate a stable cell line secreting soluble TNF-R1 (sTNF-R1). The sTNF-R1 was purified, and its biochemical properties and its interactions with human TNF-alpha were examined. SDS-PAGE resolved the purified sTNF-R1 into three bands of approximate Mr 24,200, 28,200, and 32,800. Sedimentation equilibrium analysis gave a molecular weight of 25,000 for sTNF-R1 whereas the molecular weight obtained by gel filtration chromatography was approximately 55,000-60,000. Scatchard analysis of [125I]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (Kd = 93 pM), comparable to that observed for the intact receptor on whole cells. Competitive binding experiments showed that sTNF-R1 has a 50-60-fold higher affinity for TNF-alpha than for TNF-beta, in contrast to the equal affinities of TNF-alpha and TNF-beta for the full-length TNF-R1 transiently expressed in mammalian cells. The sTNF-R1 was found to block the cytotoxicity of TNF-alpha and TNF-beta on a murine L-M cell assay. The sizes of the sTNF-R1.TNF-alpha complex determined by gel filtration chromatography and sedimentation equilibrium were approximately 141 and 115 kDa, respectively. The stoichiometry of the complex was examined by Scatchard analysis, size-exclusion chromatography, HPLC separation, amino acid composition, sequence analysis, and sedimentation equilibrium. The data from these studies suggest that at least two molecules of sTNF-R1 can bind to a single TNF-alpha trimer.</description><subject>550201 - Biochemistry- Tracer Techniques</subject><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>BASIC BIOLOGICAL SCIENCES</subject><subject>BETA DECAY RADIOISOTOPES</subject><subject>BIOCHEMICAL REACTION KINETICS</subject><subject>Biological and medical sciences</subject><subject>CELL CONSTITUENTS</subject><subject>Cell Line</subject><subject>Cell receptors</subject><subject>Cell structures and functions</subject><subject>Chromatography, Gel</subject><subject>Cytotoxicity Tests, Immunologic</subject><subject>DAYS LIVING RADIOISOTOPES</subject><subject>DISEASES</subject><subject>ELECTRON CAPTURE RADIOISOTOPES</subject><subject>ELECTROPHORESIS</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>GROWTH FACTORS</subject><subject>Hormone receptors. Growth factor receptors. Cytokine receptors. Prostaglandin receptors</subject><subject>Humans</subject><subject>INTERMEDIATE MASS NUCLEI</subject><subject>INTERNAL CONVERSION RADIOISOTOPES</subject><subject>IODINE 125</subject><subject>IODINE ISOTOPES</subject><subject>ISOTOPE APPLICATIONS</subject><subject>ISOTOPES</subject><subject>KINETICS</subject><subject>LYMPHOKINES</subject><subject>Mice</subject><subject>MITOGENS</subject><subject>Molecular and cellular biology</subject><subject>Molecular Weight</subject><subject>NECROSIS</subject><subject>NEOPLASMS</subject><subject>NUCLEI</subject><subject>ODD-EVEN NUCLEI</subject><subject>ORGANIC COMPOUNDS</subject><subject>PATHOLOGICAL CHANGES</subject><subject>PLASMIDS</subject><subject>PROTEINS</subject><subject>RADIOISOTOPES</subject><subject>RADIORECEPTOR ASSAY</subject><subject>REACTION KINETICS</subject><subject>Receptor Aggregation</subject><subject>Receptors, Cell Surface - chemistry</subject><subject>Receptors, Cell Surface - drug effects</subject><subject>Receptors, Cell Surface - isolation & purification</subject><subject>Receptors, Tumor Necrosis Factor</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - drug effects</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>signal transduction</subject><subject>Solubility</subject><subject>TRACER TECHNIQUES</subject><subject>tumor necrosis factor</subject><subject>Tumor Necrosis Factor-alpha - pharmacology</subject><subject>Ultracentrifugation</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1992</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpt0Utv1DAQAOAIgcpSOHFGshCCQ5XFdpwXN7SiFKkIJEo5WmN7suuSxFvbQS2_iR-Jt1ktHHryYz6PxjNZ9pzRJaOcvVWWUsZaoLx4kC1YyWku2rZ8mC0opVXO24o-zp6EcJWOgtbiKDtiBaOC00X2Z7UBDzqit78hWjcS1xEgHrUblB1hjARvYhLY91MPnhg3gL1TcYMk3m6RMBKnwXkyovYu2EC6lDCdUxLcps07gr-swVEj6dL1_XgJ_XYDS2JHM2k0h8cE1muP67vanmaPOugDPtuvx9n30w8Xq7P8_MvHT6v35zkIwWLOTY0llKaARinVKF4UlVCqNJWCjnZ16kEh6oa1HeVcVaXpuOKmYJrWrW7BFMfZyzmvC9HKoG1EvdFuTDVHWYqmqesiodcz2np3PWGIcrBh1yYY0U1BsopVbc3KBE9muPtw8NjJrbcD-FvJqNwNUP43wKRf7NNOakDzz84TS_FX-zgEDX3nYdQ2HFjJK8GaJrF8ZjZEvDmEwf-UVV3Upbz4-k3-uKTic9Weysvk38wedJBXbvJjavC9Bf4FlgXBbg</recordid><startdate>19920204</startdate><enddate>19920204</enddate><creator>Pennica, Diane</creator><creator>Kohr, William J</creator><creator>Fendly, Brian M</creator><creator>Shire, Steven J</creator><creator>Raab, Helga E</creator><creator>Borchardt, Paul E</creator><creator>Lewis, Martyn</creator><creator>Goeddel, David V</creator><general>American Chemical Society</general><scope>BSCLL</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>8FD</scope><scope>FR3</scope><scope>M7Z</scope><scope>P64</scope><scope>OTOTI</scope></search><sort><creationdate>19920204</creationdate><title>Characterization of a recombinant extracellular domain of the type 1 tumor necrosis factor receptor: evidence for tumor necrosis factor .alpha. induced receptor aggregation</title><author>Pennica, Diane ; Kohr, William J ; Fendly, Brian M ; Shire, Steven J ; Raab, Helga E ; Borchardt, Paul E ; Lewis, Martyn ; Goeddel, David V</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a441t-2d7e5a5d3a8bbb8b23364bb5d6baf0f7407347819f022b65df2b2d31c079c9ad3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1992</creationdate><topic>550201 - Biochemistry- Tracer Techniques</topic><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>BASIC BIOLOGICAL SCIENCES</topic><topic>BETA DECAY RADIOISOTOPES</topic><topic>BIOCHEMICAL REACTION KINETICS</topic><topic>Biological and medical sciences</topic><topic>CELL CONSTITUENTS</topic><topic>Cell Line</topic><topic>Cell receptors</topic><topic>Cell structures and functions</topic><topic>Chromatography, Gel</topic><topic>Cytotoxicity Tests, Immunologic</topic><topic>DAYS LIVING RADIOISOTOPES</topic><topic>DISEASES</topic><topic>ELECTRON CAPTURE RADIOISOTOPES</topic><topic>ELECTROPHORESIS</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>GROWTH FACTORS</topic><topic>Hormone receptors. Growth factor receptors. Cytokine receptors. Prostaglandin receptors</topic><topic>Humans</topic><topic>INTERMEDIATE MASS NUCLEI</topic><topic>INTERNAL CONVERSION RADIOISOTOPES</topic><topic>IODINE 125</topic><topic>IODINE ISOTOPES</topic><topic>ISOTOPE APPLICATIONS</topic><topic>ISOTOPES</topic><topic>KINETICS</topic><topic>LYMPHOKINES</topic><topic>Mice</topic><topic>MITOGENS</topic><topic>Molecular and cellular biology</topic><topic>Molecular Weight</topic><topic>NECROSIS</topic><topic>NEOPLASMS</topic><topic>NUCLEI</topic><topic>ODD-EVEN NUCLEI</topic><topic>ORGANIC COMPOUNDS</topic><topic>PATHOLOGICAL CHANGES</topic><topic>PLASMIDS</topic><topic>PROTEINS</topic><topic>RADIOISOTOPES</topic><topic>RADIORECEPTOR ASSAY</topic><topic>REACTION KINETICS</topic><topic>Receptor Aggregation</topic><topic>Receptors, Cell Surface - chemistry</topic><topic>Receptors, Cell Surface - drug effects</topic><topic>Receptors, Cell Surface - isolation & purification</topic><topic>Receptors, Tumor Necrosis Factor</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - drug effects</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>signal transduction</topic><topic>Solubility</topic><topic>TRACER TECHNIQUES</topic><topic>tumor necrosis factor</topic><topic>Tumor Necrosis Factor-alpha - pharmacology</topic><topic>Ultracentrifugation</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Pennica, Diane</creatorcontrib><creatorcontrib>Kohr, William J</creatorcontrib><creatorcontrib>Fendly, Brian M</creatorcontrib><creatorcontrib>Shire, Steven J</creatorcontrib><creatorcontrib>Raab, Helga E</creatorcontrib><creatorcontrib>Borchardt, Paul E</creatorcontrib><creatorcontrib>Lewis, Martyn</creatorcontrib><creatorcontrib>Goeddel, David V</creatorcontrib><collection>Istex</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>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biochemistry Abstracts 1</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>OSTI.GOV</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Pennica, Diane</au><au>Kohr, William J</au><au>Fendly, Brian M</au><au>Shire, Steven J</au><au>Raab, Helga E</au><au>Borchardt, Paul E</au><au>Lewis, Martyn</au><au>Goeddel, David V</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Characterization of a recombinant extracellular domain of the type 1 tumor necrosis factor receptor: evidence for tumor necrosis factor .alpha. induced receptor aggregation</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>1992-02-04</date><risdate>1992</risdate><volume>31</volume><issue>4</issue><spage>1134</spage><epage>1141</epage><pages>1134-1141</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>An expression plasmid encoding the extracellular portion of the human tumor necrosis factor (TNF) type 1 receptor (TNF-R1) was constructed and used to generate a stable cell line secreting soluble TNF-R1 (sTNF-R1). The sTNF-R1 was purified, and its biochemical properties and its interactions with human TNF-alpha were examined. SDS-PAGE resolved the purified sTNF-R1 into three bands of approximate Mr 24,200, 28,200, and 32,800. Sedimentation equilibrium analysis gave a molecular weight of 25,000 for sTNF-R1 whereas the molecular weight obtained by gel filtration chromatography was approximately 55,000-60,000. Scatchard analysis of [125I]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (Kd = 93 pM), comparable to that observed for the intact receptor on whole cells. Competitive binding experiments showed that sTNF-R1 has a 50-60-fold higher affinity for TNF-alpha than for TNF-beta, in contrast to the equal affinities of TNF-alpha and TNF-beta for the full-length TNF-R1 transiently expressed in mammalian cells. The sTNF-R1 was found to block the cytotoxicity of TNF-alpha and TNF-beta on a murine L-M cell assay. The sizes of the sTNF-R1.TNF-alpha complex determined by gel filtration chromatography and sedimentation equilibrium were approximately 141 and 115 kDa, respectively. The stoichiometry of the complex was examined by Scatchard analysis, size-exclusion chromatography, HPLC separation, amino acid composition, sequence analysis, and sedimentation equilibrium. The data from these studies suggest that at least two molecules of sTNF-R1 can bind to a single TNF-alpha trimer.</abstract><cop>Washington, DC</cop><pub>American Chemical Society</pub><pmid>1310420</pmid><doi>10.1021/bi00119a023</doi><tpages>8</tpages></addata></record> |
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| ispartof | Biochemistry (Easton), 1992-02, Vol.31 (4), p.1134-1141 |
| issn | 0006-2960 1520-4995 |
| language | eng |
| recordid | cdi_osti_scitechconnect_5488773 |
| source | MEDLINE; ACS Publications |
| subjects | 550201 - Biochemistry- Tracer Techniques Amino Acid Sequence Animals BASIC BIOLOGICAL SCIENCES BETA DECAY RADIOISOTOPES BIOCHEMICAL REACTION KINETICS Biological and medical sciences CELL CONSTITUENTS Cell Line Cell receptors Cell structures and functions Chromatography, Gel Cytotoxicity Tests, Immunologic DAYS LIVING RADIOISOTOPES DISEASES ELECTRON CAPTURE RADIOISOTOPES ELECTROPHORESIS Fundamental and applied biological sciences. Psychology GROWTH FACTORS Hormone receptors. Growth factor receptors. Cytokine receptors. Prostaglandin receptors Humans INTERMEDIATE MASS NUCLEI INTERNAL CONVERSION RADIOISOTOPES IODINE 125 IODINE ISOTOPES ISOTOPE APPLICATIONS ISOTOPES KINETICS LYMPHOKINES Mice MITOGENS Molecular and cellular biology Molecular Weight NECROSIS NEOPLASMS NUCLEI ODD-EVEN NUCLEI ORGANIC COMPOUNDS PATHOLOGICAL CHANGES PLASMIDS PROTEINS RADIOISOTOPES RADIORECEPTOR ASSAY REACTION KINETICS Receptor Aggregation Receptors, Cell Surface - chemistry Receptors, Cell Surface - drug effects Receptors, Cell Surface - isolation & purification Receptors, Tumor Necrosis Factor Recombinant Proteins - chemistry Recombinant Proteins - drug effects Recombinant Proteins - isolation & purification signal transduction Solubility TRACER TECHNIQUES tumor necrosis factor Tumor Necrosis Factor-alpha - pharmacology Ultracentrifugation |
| title | Characterization of a recombinant extracellular domain of the type 1 tumor necrosis factor receptor: evidence for tumor necrosis factor .alpha. induced receptor aggregation |
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