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Interaction of Fibronectin With Cultured Human Endothelial Cells: Characterization of the Specific Receptor
In this study we provide a characterization of the fibronectin (FN) binding to endothelial cells (EC), and we identify the FN binding site on these cells. 125I-FN binding to EC in suspension was time dependent and reached a plateau at 4 h. Cold FN inhibited this interaction in a concentration-depend...
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Published in: | Blood 1989-05, Vol.73 (6), p.1576-1585 |
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container_title | Blood |
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creator | Conforti, Grazia Zanetti, Adriana Colella, Silvia Abbadini, Marzia Marchisio, Pier Carlo Pytela, Robert Giancotti, Filippo Tarone, Guido Languino, Lucia Raffaella Dejana, Elisabetta |
description | In this study we provide a characterization of the fibronectin (FN) binding to endothelial cells (EC), and we identify the FN binding site on these cells. 125I-FN binding to EC in suspension was time dependent and reached a plateau at 4 h. Cold FN inhibited this interaction in a concentration-dependent way, but vitronectin, fibrinogen, and IgG were poorly effective. About 80% of the total FN associated to EC at the equilibrium was specifically bound; of this, 60% was reversibly bound, while 20% appeared to be internalized. The FN binding was saturable and an apparent dissociation constant of about 0.23 � 10-6 mol/L and a maximal number of binding sites of about 9.8 � 105 was estimated from binding isotherms. Autoradiography data showed that EC-associated 125I-FN was all in high mol wt form that did not enter the gel. We then characterize the FN receptor (FNR) in EC. An antiserum to the FNR isolated from human placenta inhibited FN binding to EC by 89%, and using the immunoblotting technique, it recognized two bands in the EC detergent extract of mol wt 125/160 Kd. This antiserum also recognized the EC membrane protein complex eluted from the FN affinity column by an arg-gly-asp (RGD) peptide. When this complex was included into liposomes, it poorly bound to FN. However, the binding was strikingly increased by addition of Mn in the buffer and was specific for FN in respect to other substrata. These data define the FN binding site in EC and indicate that it is functionally and structurally related to that isolated from human placenta. |
doi_str_mv | 10.1182/blood.V73.6.1576.1576 |
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Cold FN inhibited this interaction in a concentration-dependent way, but vitronectin, fibrinogen, and IgG were poorly effective. About 80% of the total FN associated to EC at the equilibrium was specifically bound; of this, 60% was reversibly bound, while 20% appeared to be internalized. The FN binding was saturable and an apparent dissociation constant of about 0.23 � 10-6 mol/L and a maximal number of binding sites of about 9.8 � 105 was estimated from binding isotherms. Autoradiography data showed that EC-associated 125I-FN was all in high mol wt form that did not enter the gel. We then characterize the FN receptor (FNR) in EC. An antiserum to the FNR isolated from human placenta inhibited FN binding to EC by 89%, and using the immunoblotting technique, it recognized two bands in the EC detergent extract of mol wt 125/160 Kd. This antiserum also recognized the EC membrane protein complex eluted from the FN affinity column by an arg-gly-asp (RGD) peptide. When this complex was included into liposomes, it poorly bound to FN. However, the binding was strikingly increased by addition of Mn in the buffer and was specific for FN in respect to other substrata. These data define the FN binding site in EC and indicate that it is functionally and structurally related to that isolated from human placenta.</description><identifier>ISSN: 0006-4971</identifier><identifier>EISSN: 1528-0020</identifier><identifier>DOI: 10.1182/blood.V73.6.1576.1576</identifier><identifier>PMID: 2469496</identifier><language>eng</language><publisher>Washington, DC: Elsevier Inc</publisher><subject>Analytical, structural and metabolic biochemistry ; Antigens, Surface ; Biological and medical sciences ; Cell Adhesion Molecules ; Cells, Cultured ; Endothelium, Vascular - physiology ; Extracellular Matrix - metabolism ; Fibrinogen - metabolism ; Fibronectins - physiology ; Fundamental and applied biological sciences. Psychology ; Glycoproteins ; Glycoproteins - metabolism ; Humans ; In Vitro Techniques ; Kinetics ; Liposomes ; Molecular Weight ; Proteins ; Receptors, Fibronectin ; Receptors, Immunologic - physiology ; Vitronectin</subject><ispartof>Blood, 1989-05, Vol.73 (6), p.1576-1585</ispartof><rights>1989 American Society of Hematology</rights><rights>1990 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c436t-c9f499fdfcb3e3370eb08df0696d19f5bcc3f7f68f5e351c2ed09837d14feb4b3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0006497120789603$$EHTML$$P50$$Gelsevier$$Hfree_for_read</linktohtml><link.rule.ids>314,780,784,3549,27924,27925,45780</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=6839722$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2469496$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Conforti, Grazia</creatorcontrib><creatorcontrib>Zanetti, Adriana</creatorcontrib><creatorcontrib>Colella, Silvia</creatorcontrib><creatorcontrib>Abbadini, Marzia</creatorcontrib><creatorcontrib>Marchisio, Pier Carlo</creatorcontrib><creatorcontrib>Pytela, Robert</creatorcontrib><creatorcontrib>Giancotti, Filippo</creatorcontrib><creatorcontrib>Tarone, Guido</creatorcontrib><creatorcontrib>Languino, Lucia Raffaella</creatorcontrib><creatorcontrib>Dejana, Elisabetta</creatorcontrib><title>Interaction of Fibronectin With Cultured Human Endothelial Cells: Characterization of the Specific Receptor</title><title>Blood</title><addtitle>Blood</addtitle><description>In this study we provide a characterization of the fibronectin (FN) binding to endothelial cells (EC), and we identify the FN binding site on these cells. 125I-FN binding to EC in suspension was time dependent and reached a plateau at 4 h. Cold FN inhibited this interaction in a concentration-dependent way, but vitronectin, fibrinogen, and IgG were poorly effective. About 80% of the total FN associated to EC at the equilibrium was specifically bound; of this, 60% was reversibly bound, while 20% appeared to be internalized. The FN binding was saturable and an apparent dissociation constant of about 0.23 � 10-6 mol/L and a maximal number of binding sites of about 9.8 � 105 was estimated from binding isotherms. Autoradiography data showed that EC-associated 125I-FN was all in high mol wt form that did not enter the gel. We then characterize the FN receptor (FNR) in EC. An antiserum to the FNR isolated from human placenta inhibited FN binding to EC by 89%, and using the immunoblotting technique, it recognized two bands in the EC detergent extract of mol wt 125/160 Kd. This antiserum also recognized the EC membrane protein complex eluted from the FN affinity column by an arg-gly-asp (RGD) peptide. When this complex was included into liposomes, it poorly bound to FN. However, the binding was strikingly increased by addition of Mn in the buffer and was specific for FN in respect to other substrata. These data define the FN binding site in EC and indicate that it is functionally and structurally related to that isolated from human placenta.</description><subject>Analytical, structural and metabolic biochemistry</subject><subject>Antigens, Surface</subject><subject>Biological and medical sciences</subject><subject>Cell Adhesion Molecules</subject><subject>Cells, Cultured</subject><subject>Endothelium, Vascular - physiology</subject><subject>Extracellular Matrix - metabolism</subject><subject>Fibrinogen - metabolism</subject><subject>Fibronectins - physiology</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Glycoproteins</subject><subject>Glycoproteins - metabolism</subject><subject>Humans</subject><subject>In Vitro Techniques</subject><subject>Kinetics</subject><subject>Liposomes</subject><subject>Molecular Weight</subject><subject>Proteins</subject><subject>Receptors, Fibronectin</subject><subject>Receptors, Immunologic - physiology</subject><subject>Vitronectin</subject><issn>0006-4971</issn><issn>1528-0020</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1989</creationdate><recordtype>article</recordtype><recordid>eNqFkUFvFCEUx4nR1G31IzThYLzNFgYGBi_GTFrbpEkTW_VIGHhk0VlYYcakfnrZ7tqrl0fI-70_Lz8QOqdkTWnfXoxTSm79TbK1WNNOHsoLtKJd2zeEtOQlWhFCRMOVpK_RaSk_CKGctd0JOmm5UFyJFfp5E2fIxs4hRZw8vgpjThHqPeLvYd7gYZnmJYPD18vWRHwZXZo3MAUz4QGmqXzAw8bsAyCHP-ZfTEXw_Q5s8MHiL2BhN6f8Br3yZirw9nieoa9Xlw_DdXN79_lm-HTbWM7E3FjluVLeeTsyYEwSGEnvPBFKOKp8N1rLvPSi9x2wjtoWHFE9k45yDyMf2Rl6f8jd5fRrgTLrbSi2LmsipKVo2StJuKIV7A6gzamUDF7vctia_Kgp0XvJ-kmyrpK10Hu_T6XOnR8fWMYtuOepo9Xaf3fsm2LN5LOJNpRnTPRMybat2McDBlXG7wBZFxsgWnAh1x_QLoX_LPIXU8Gd9Q</recordid><startdate>19890501</startdate><enddate>19890501</enddate><creator>Conforti, Grazia</creator><creator>Zanetti, Adriana</creator><creator>Colella, Silvia</creator><creator>Abbadini, Marzia</creator><creator>Marchisio, Pier Carlo</creator><creator>Pytela, Robert</creator><creator>Giancotti, Filippo</creator><creator>Tarone, Guido</creator><creator>Languino, Lucia Raffaella</creator><creator>Dejana, Elisabetta</creator><general>Elsevier Inc</general><general>The Americain Society of Hematology</general><scope>6I.</scope><scope>AAFTH</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></search><sort><creationdate>19890501</creationdate><title>Interaction of Fibronectin With Cultured Human Endothelial Cells: Characterization of the Specific Receptor</title><author>Conforti, Grazia ; Zanetti, Adriana ; Colella, Silvia ; Abbadini, Marzia ; Marchisio, Pier Carlo ; Pytela, Robert ; Giancotti, Filippo ; Tarone, Guido ; Languino, Lucia Raffaella ; Dejana, Elisabetta</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c436t-c9f499fdfcb3e3370eb08df0696d19f5bcc3f7f68f5e351c2ed09837d14feb4b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1989</creationdate><topic>Analytical, structural and metabolic biochemistry</topic><topic>Antigens, Surface</topic><topic>Biological and medical sciences</topic><topic>Cell Adhesion Molecules</topic><topic>Cells, Cultured</topic><topic>Endothelium, Vascular - physiology</topic><topic>Extracellular Matrix - metabolism</topic><topic>Fibrinogen - metabolism</topic><topic>Fibronectins - physiology</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Glycoproteins</topic><topic>Glycoproteins - metabolism</topic><topic>Humans</topic><topic>In Vitro Techniques</topic><topic>Kinetics</topic><topic>Liposomes</topic><topic>Molecular Weight</topic><topic>Proteins</topic><topic>Receptors, Fibronectin</topic><topic>Receptors, Immunologic - physiology</topic><topic>Vitronectin</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Conforti, Grazia</creatorcontrib><creatorcontrib>Zanetti, Adriana</creatorcontrib><creatorcontrib>Colella, Silvia</creatorcontrib><creatorcontrib>Abbadini, Marzia</creatorcontrib><creatorcontrib>Marchisio, Pier Carlo</creatorcontrib><creatorcontrib>Pytela, Robert</creatorcontrib><creatorcontrib>Giancotti, Filippo</creatorcontrib><creatorcontrib>Tarone, Guido</creatorcontrib><creatorcontrib>Languino, Lucia Raffaella</creatorcontrib><creatorcontrib>Dejana, Elisabetta</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</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><jtitle>Blood</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Conforti, Grazia</au><au>Zanetti, Adriana</au><au>Colella, Silvia</au><au>Abbadini, Marzia</au><au>Marchisio, Pier Carlo</au><au>Pytela, Robert</au><au>Giancotti, Filippo</au><au>Tarone, Guido</au><au>Languino, Lucia Raffaella</au><au>Dejana, Elisabetta</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Interaction of Fibronectin With Cultured Human Endothelial Cells: Characterization of the Specific Receptor</atitle><jtitle>Blood</jtitle><addtitle>Blood</addtitle><date>1989-05-01</date><risdate>1989</risdate><volume>73</volume><issue>6</issue><spage>1576</spage><epage>1585</epage><pages>1576-1585</pages><issn>0006-4971</issn><eissn>1528-0020</eissn><abstract>In this study we provide a characterization of the fibronectin (FN) binding to endothelial cells (EC), and we identify the FN binding site on these cells. 125I-FN binding to EC in suspension was time dependent and reached a plateau at 4 h. Cold FN inhibited this interaction in a concentration-dependent way, but vitronectin, fibrinogen, and IgG were poorly effective. About 80% of the total FN associated to EC at the equilibrium was specifically bound; of this, 60% was reversibly bound, while 20% appeared to be internalized. The FN binding was saturable and an apparent dissociation constant of about 0.23 � 10-6 mol/L and a maximal number of binding sites of about 9.8 � 105 was estimated from binding isotherms. Autoradiography data showed that EC-associated 125I-FN was all in high mol wt form that did not enter the gel. We then characterize the FN receptor (FNR) in EC. An antiserum to the FNR isolated from human placenta inhibited FN binding to EC by 89%, and using the immunoblotting technique, it recognized two bands in the EC detergent extract of mol wt 125/160 Kd. This antiserum also recognized the EC membrane protein complex eluted from the FN affinity column by an arg-gly-asp (RGD) peptide. When this complex was included into liposomes, it poorly bound to FN. However, the binding was strikingly increased by addition of Mn in the buffer and was specific for FN in respect to other substrata. These data define the FN binding site in EC and indicate that it is functionally and structurally related to that isolated from human placenta.</abstract><cop>Washington, DC</cop><pub>Elsevier Inc</pub><pmid>2469496</pmid><doi>10.1182/blood.V73.6.1576.1576</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Analytical, structural and metabolic biochemistry Antigens, Surface Biological and medical sciences Cell Adhesion Molecules Cells, Cultured Endothelium, Vascular - physiology Extracellular Matrix - metabolism Fibrinogen - metabolism Fibronectins - physiology Fundamental and applied biological sciences. Psychology Glycoproteins Glycoproteins - metabolism Humans In Vitro Techniques Kinetics Liposomes Molecular Weight Proteins Receptors, Fibronectin Receptors, Immunologic - physiology Vitronectin |
title | Interaction of Fibronectin With Cultured Human Endothelial Cells: Characterization of the Specific Receptor |
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