The role of Nedd4-1 WW domains in binding and regulating human organic anion transporter 1
Human organic anion transporter 1 (hOAT1), expressed at the basolateral membrane of kidney proximal tubule cells, mediates the active renal secretion of a diverse array of clinically important drugs, including anti-human immunodeficiency virus therapeutics, antitumor drugs, antibiotics, antihyperten...
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Veröffentlicht in: | American journal of physiology. Renal physiology 2016-08, Vol.311 (2), p.F320-F329 |
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description | Human organic anion transporter 1 (hOAT1), expressed at the basolateral membrane of kidney proximal tubule cells, mediates the active renal secretion of a diverse array of clinically important drugs, including anti-human immunodeficiency virus therapeutics, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We have previously demonstrated that posttranslational modification of hOAT1 by ubiquitination is an important mechanism for the regulation of this transporter. The present study aimed at identifying the ubiquitin ligase for hOAT1 and its mechanism of action. We showed that overexpression of neural precursor cell expressed, developmentally downregulated (Nedd)4-1, an E3 ubiquitin ligase, enhanced hOAT1 ubiquitination, decreased hOAT1 expression at the cell surface, and inhibited hOAT1 transport activity. In contrast, overexpression of the ubiquitin ligase-dead mutant Nedd4-1/C867S was without effects on hOAT1. Furthermore, knockdown of endogenously expressed Nedd4-1 by Nedd4-1-specific small interfering RNA reduced hOAT1 ubiquitination. Immunoprecipitation experiments in cultured cells and rat kidney slices and immunofluorescence experiments in rat kidney slices showed that there was a physical interaction between OAT1 and Nedd4-1. Nedd4-1 contains four protein-protein interacting WW domains. When these WW domains were inactivated by mutating two amino acid residues in each of the four WW domains (Mut-WW1: V210W/H212G, Mut-WW2: V367W/H369G, Mut-WW3: I440W/H442G, and Mut-WW4: I492W/H494G, respectively), only Mut-WW2 and Mut-WW3 significantly lost their ability to bind and to ubiquitinate hOAT1. As a result, Mut-WW2 and Mut-WW3 were unable to suppress hOAT1-mediated transport as effectively as wild-type Nedd4-1. In conclusion, this is the first demonstration that Nedd4-1 regulates hOAT1 ubiquitination, expression, and transport activity through its WW2 and WW3 domains. |
doi_str_mv | 10.1152/ajprenal.00153.2016 |
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We have previously demonstrated that posttranslational modification of hOAT1 by ubiquitination is an important mechanism for the regulation of this transporter. The present study aimed at identifying the ubiquitin ligase for hOAT1 and its mechanism of action. We showed that overexpression of neural precursor cell expressed, developmentally downregulated (Nedd)4-1, an E3 ubiquitin ligase, enhanced hOAT1 ubiquitination, decreased hOAT1 expression at the cell surface, and inhibited hOAT1 transport activity. In contrast, overexpression of the ubiquitin ligase-dead mutant Nedd4-1/C867S was without effects on hOAT1. Furthermore, knockdown of endogenously expressed Nedd4-1 by Nedd4-1-specific small interfering RNA reduced hOAT1 ubiquitination. Immunoprecipitation experiments in cultured cells and rat kidney slices and immunofluorescence experiments in rat kidney slices showed that there was a physical interaction between OAT1 and Nedd4-1. Nedd4-1 contains four protein-protein interacting WW domains. When these WW domains were inactivated by mutating two amino acid residues in each of the four WW domains (Mut-WW1: V210W/H212G, Mut-WW2: V367W/H369G, Mut-WW3: I440W/H442G, and Mut-WW4: I492W/H494G, respectively), only Mut-WW2 and Mut-WW3 significantly lost their ability to bind and to ubiquitinate hOAT1. As a result, Mut-WW2 and Mut-WW3 were unable to suppress hOAT1-mediated transport as effectively as wild-type Nedd4-1. In conclusion, this is the first demonstration that Nedd4-1 regulates hOAT1 ubiquitination, expression, and transport activity through its WW2 and WW3 domains.</description><identifier>ISSN: 1931-857X</identifier><identifier>EISSN: 1522-1466</identifier><identifier>DOI: 10.1152/ajprenal.00153.2016</identifier><identifier>PMID: 27226107</identifier><language>eng</language><publisher>United States: American Physiological Society</publisher><subject>Animals ; Binding sites ; Biotinylation ; Cells ; Cercopithecus aethiops ; COS Cells ; Endosomal Sorting Complexes Required for Transport - genetics ; Endosomal Sorting Complexes Required for Transport - metabolism ; Experiments ; HEK293 Cells ; Humans ; In Vitro Techniques ; Kidney - metabolism ; Kidneys ; Membrane Proteins - metabolism ; Mutagenesis, Site-Directed ; Nedd4 Ubiquitin Protein Ligases ; Organic Anion Transport Protein 1 - genetics ; Organic Anion Transport Protein 1 - metabolism ; Protein Binding ; Protein Processing, Post-Translational ; Proteins ; Rats ; Rats, Sprague-Dawley ; Ubiquitin-Protein Ligases - genetics ; Ubiquitin-Protein Ligases - metabolism ; Ubiquitination</subject><ispartof>American journal of physiology. Renal physiology, 2016-08, Vol.311 (2), p.F320-F329</ispartof><rights>Copyright © 2016 the American Physiological Society.</rights><rights>Copyright American Physiological Society Aug 1, 2016</rights><rights>Copyright © 2016 the American Physiological Society 2016 American Physiological Society</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c466t-30076544e5b26a068d6d25f181b64057cc1a0a471c4e0ca66442604584f8b8e33</citedby><cites>FETCH-LOGICAL-c466t-30076544e5b26a068d6d25f181b64057cc1a0a471c4e0ca66442604584f8b8e33</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,776,780,881,3026,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27226107$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Xu, Da</creatorcontrib><creatorcontrib>Wang, Haoxun</creatorcontrib><creatorcontrib>Gardner, Carol</creatorcontrib><creatorcontrib>Pan, Zui</creatorcontrib><creatorcontrib>Zhang, Ping L</creatorcontrib><creatorcontrib>Zhang, Jinghui</creatorcontrib><creatorcontrib>You, Guofeng</creatorcontrib><title>The role of Nedd4-1 WW domains in binding and regulating human organic anion transporter 1</title><title>American journal of physiology. Renal physiology</title><addtitle>Am J Physiol Renal Physiol</addtitle><description>Human organic anion transporter 1 (hOAT1), expressed at the basolateral membrane of kidney proximal tubule cells, mediates the active renal secretion of a diverse array of clinically important drugs, including anti-human immunodeficiency virus therapeutics, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We have previously demonstrated that posttranslational modification of hOAT1 by ubiquitination is an important mechanism for the regulation of this transporter. The present study aimed at identifying the ubiquitin ligase for hOAT1 and its mechanism of action. We showed that overexpression of neural precursor cell expressed, developmentally downregulated (Nedd)4-1, an E3 ubiquitin ligase, enhanced hOAT1 ubiquitination, decreased hOAT1 expression at the cell surface, and inhibited hOAT1 transport activity. In contrast, overexpression of the ubiquitin ligase-dead mutant Nedd4-1/C867S was without effects on hOAT1. Furthermore, knockdown of endogenously expressed Nedd4-1 by Nedd4-1-specific small interfering RNA reduced hOAT1 ubiquitination. Immunoprecipitation experiments in cultured cells and rat kidney slices and immunofluorescence experiments in rat kidney slices showed that there was a physical interaction between OAT1 and Nedd4-1. Nedd4-1 contains four protein-protein interacting WW domains. When these WW domains were inactivated by mutating two amino acid residues in each of the four WW domains (Mut-WW1: V210W/H212G, Mut-WW2: V367W/H369G, Mut-WW3: I440W/H442G, and Mut-WW4: I492W/H494G, respectively), only Mut-WW2 and Mut-WW3 significantly lost their ability to bind and to ubiquitinate hOAT1. As a result, Mut-WW2 and Mut-WW3 were unable to suppress hOAT1-mediated transport as effectively as wild-type Nedd4-1. In conclusion, this is the first demonstration that Nedd4-1 regulates hOAT1 ubiquitination, expression, and transport activity through its WW2 and WW3 domains.</description><subject>Animals</subject><subject>Binding sites</subject><subject>Biotinylation</subject><subject>Cells</subject><subject>Cercopithecus aethiops</subject><subject>COS Cells</subject><subject>Endosomal Sorting Complexes Required for Transport - genetics</subject><subject>Endosomal Sorting Complexes Required for Transport - metabolism</subject><subject>Experiments</subject><subject>HEK293 Cells</subject><subject>Humans</subject><subject>In Vitro Techniques</subject><subject>Kidney - metabolism</subject><subject>Kidneys</subject><subject>Membrane Proteins - metabolism</subject><subject>Mutagenesis, Site-Directed</subject><subject>Nedd4 Ubiquitin Protein Ligases</subject><subject>Organic Anion Transport Protein 1 - genetics</subject><subject>Organic Anion Transport Protein 1 - metabolism</subject><subject>Protein Binding</subject><subject>Protein Processing, Post-Translational</subject><subject>Proteins</subject><subject>Rats</subject><subject>Rats, Sprague-Dawley</subject><subject>Ubiquitin-Protein Ligases - genetics</subject><subject>Ubiquitin-Protein Ligases - metabolism</subject><subject>Ubiquitination</subject><issn>1931-857X</issn><issn>1522-1466</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpdkc1qGzEUhUVpaBw3T1Aogm66GUdXfzPeFErIH4Rk4-CSjdBoNLbMjORKM4G-fWQnDmlWkjjfvRzxIfQNyAxA0DO92UbrdTcjBASbUQLyE5rkhBbApfyc73MGRSXKP8foJKUNySBQ-IKOaUmpBFJO0ONibXEMncWhxXe2aXgBeLnETei18wk7j2vnG-dXWPsGR7saOz3snuux1x6HuNLemRy64PEQtU_bEAcbMXxFR63ukj19Pafo4fJicX5d3N5f3Zz_vi1MbjkUjJBSCs6tqKnURFaNbKhooYJaciJKY0ATzUsw3BKjpeScSsJFxduqrixjU_TrZe92rHvbGOtzjU5to-t1_KeCdur_xLu1WoUnJShnlEJe8PN1QQx_R5sG1btkbNdpb8OYVK4i5BwYlRn98QHdhDFmCXsKaDUXoswUe6FMDClF276VAaJ27tTBndq7Uzt3eer7-3-8zRxksWfnPZW_</recordid><startdate>20160801</startdate><enddate>20160801</enddate><creator>Xu, Da</creator><creator>Wang, Haoxun</creator><creator>Gardner, Carol</creator><creator>Pan, Zui</creator><creator>Zhang, Ping L</creator><creator>Zhang, Jinghui</creator><creator>You, Guofeng</creator><general>American Physiological Society</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>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>5PM</scope></search><sort><creationdate>20160801</creationdate><title>The role of Nedd4-1 WW domains in binding and regulating human organic anion transporter 1</title><author>Xu, Da ; Wang, Haoxun ; Gardner, Carol ; Pan, Zui ; Zhang, Ping L ; Zhang, Jinghui ; You, Guofeng</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c466t-30076544e5b26a068d6d25f181b64057cc1a0a471c4e0ca66442604584f8b8e33</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>Animals</topic><topic>Binding sites</topic><topic>Biotinylation</topic><topic>Cells</topic><topic>Cercopithecus aethiops</topic><topic>COS Cells</topic><topic>Endosomal Sorting Complexes Required for Transport - genetics</topic><topic>Endosomal Sorting Complexes Required for Transport - metabolism</topic><topic>Experiments</topic><topic>HEK293 Cells</topic><topic>Humans</topic><topic>In Vitro Techniques</topic><topic>Kidney - metabolism</topic><topic>Kidneys</topic><topic>Membrane Proteins - metabolism</topic><topic>Mutagenesis, Site-Directed</topic><topic>Nedd4 Ubiquitin Protein Ligases</topic><topic>Organic Anion Transport Protein 1 - genetics</topic><topic>Organic Anion Transport Protein 1 - metabolism</topic><topic>Protein Binding</topic><topic>Protein Processing, Post-Translational</topic><topic>Proteins</topic><topic>Rats</topic><topic>Rats, Sprague-Dawley</topic><topic>Ubiquitin-Protein Ligases - genetics</topic><topic>Ubiquitin-Protein Ligases - metabolism</topic><topic>Ubiquitination</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Xu, Da</creatorcontrib><creatorcontrib>Wang, Haoxun</creatorcontrib><creatorcontrib>Gardner, Carol</creatorcontrib><creatorcontrib>Pan, Zui</creatorcontrib><creatorcontrib>Zhang, Ping L</creatorcontrib><creatorcontrib>Zhang, Jinghui</creatorcontrib><creatorcontrib>You, Guofeng</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>American journal of physiology. Renal physiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Xu, Da</au><au>Wang, Haoxun</au><au>Gardner, Carol</au><au>Pan, Zui</au><au>Zhang, Ping L</au><au>Zhang, Jinghui</au><au>You, Guofeng</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>The role of Nedd4-1 WW domains in binding and regulating human organic anion transporter 1</atitle><jtitle>American journal of physiology. Renal physiology</jtitle><addtitle>Am J Physiol Renal Physiol</addtitle><date>2016-08-01</date><risdate>2016</risdate><volume>311</volume><issue>2</issue><spage>F320</spage><epage>F329</epage><pages>F320-F329</pages><issn>1931-857X</issn><eissn>1522-1466</eissn><abstract>Human organic anion transporter 1 (hOAT1), expressed at the basolateral membrane of kidney proximal tubule cells, mediates the active renal secretion of a diverse array of clinically important drugs, including anti-human immunodeficiency virus therapeutics, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We have previously demonstrated that posttranslational modification of hOAT1 by ubiquitination is an important mechanism for the regulation of this transporter. The present study aimed at identifying the ubiquitin ligase for hOAT1 and its mechanism of action. We showed that overexpression of neural precursor cell expressed, developmentally downregulated (Nedd)4-1, an E3 ubiquitin ligase, enhanced hOAT1 ubiquitination, decreased hOAT1 expression at the cell surface, and inhibited hOAT1 transport activity. In contrast, overexpression of the ubiquitin ligase-dead mutant Nedd4-1/C867S was without effects on hOAT1. Furthermore, knockdown of endogenously expressed Nedd4-1 by Nedd4-1-specific small interfering RNA reduced hOAT1 ubiquitination. Immunoprecipitation experiments in cultured cells and rat kidney slices and immunofluorescence experiments in rat kidney slices showed that there was a physical interaction between OAT1 and Nedd4-1. Nedd4-1 contains four protein-protein interacting WW domains. When these WW domains were inactivated by mutating two amino acid residues in each of the four WW domains (Mut-WW1: V210W/H212G, Mut-WW2: V367W/H369G, Mut-WW3: I440W/H442G, and Mut-WW4: I492W/H494G, respectively), only Mut-WW2 and Mut-WW3 significantly lost their ability to bind and to ubiquitinate hOAT1. As a result, Mut-WW2 and Mut-WW3 were unable to suppress hOAT1-mediated transport as effectively as wild-type Nedd4-1. In conclusion, this is the first demonstration that Nedd4-1 regulates hOAT1 ubiquitination, expression, and transport activity through its WW2 and WW3 domains.</abstract><cop>United States</cop><pub>American Physiological Society</pub><pmid>27226107</pmid><doi>10.1152/ajprenal.00153.2016</doi><oa>free_for_read</oa></addata></record> |
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subjects | Animals Binding sites Biotinylation Cells Cercopithecus aethiops COS Cells Endosomal Sorting Complexes Required for Transport - genetics Endosomal Sorting Complexes Required for Transport - metabolism Experiments HEK293 Cells Humans In Vitro Techniques Kidney - metabolism Kidneys Membrane Proteins - metabolism Mutagenesis, Site-Directed Nedd4 Ubiquitin Protein Ligases Organic Anion Transport Protein 1 - genetics Organic Anion Transport Protein 1 - metabolism Protein Binding Protein Processing, Post-Translational Proteins Rats Rats, Sprague-Dawley Ubiquitin-Protein Ligases - genetics Ubiquitin-Protein Ligases - metabolism Ubiquitination |
title | The role of Nedd4-1 WW domains in binding and regulating human organic anion transporter 1 |
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