Shank2E binds NaP(i) cotransporter at the apical membrane of proximal tubule cells
Proteins expressing postsynaptic density (PSD)-95/Drosophila disk large (Dlg)/zonula occludens-1 (ZO-1) (PDZ) domains are commonly involved in moderating receptor, channel, and transporter activities at the plasma membrane in a variety of cell types. At the apical membrane of renal proximal tubules...
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Veröffentlicht in: | American Journal of Physiology: Cell Physiology 2005-10, Vol.289 (4), p.C1042-C1051 |
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creator | McWilliams, Ryan R Breusegem, Sophia Y Brodsky, Kelley F Kim, Eunjoon Levi, Moshe Doctor, R Brian |
description | Proteins expressing postsynaptic density (PSD)-95/Drosophila disk large (Dlg)/zonula occludens-1 (ZO-1) (PDZ) domains are commonly involved in moderating receptor, channel, and transporter activities at the plasma membrane in a variety of cell types. At the apical membrane of renal proximal tubules (PT), the type IIa NaP(i) cotransporter (NaP(i)-IIa) binds specific PDZ domain proteins. Shank2E is a spliceoform of a family of PDZ proteins that is concentrated at the apical domain of liver and pancreatic epithelial cell types and is expressed in kidney. In the present study, immunoblotting of enriched plasma membrane fractions and immunohistology found Shank2E concentrated at the brush border membrane of rat PT cells. Confocal localization of Flag-Shank2E and enhanced green fluorescent protein-NaP(i)-IIa in cotransfected OK cells showed these proteins colocalized in the apical microvilli of this PT cell model. Shank2E co-immunoprecipitated with NaP(i)-IIa from rat renal cortex tissue and HA-NaP(i)-IIa coprecipitated with Flag-Shank2E in cotransfected human embryonic kidney HEK cells. Domain analysis showed that the PDZ domain of Shank2E specifically bound NaP(i)-IIa and truncation of the COOH-terminal TRL motif from NaP(i)-IIa abolished this binding, and Far Western blotting showed that the Shank2E- NaP(i)-IIa interaction occurred directly between the two proteins. NaP(i)-IIa activity is regulated by moderating its abundance in the apical membrane. High-P(i) conditions induce NaP(i)-IIa internalization and degradation. In both rat kidney PT cells and OK cells, shifting to high-P(i) conditions induced an acute internal redistribution of Shank2E and, in OK cells, a significant degree of degradation. In sum, Shank2E is concentrated in the apical domain of renal PT cells, specifically binds NaP(i)-IIa via PDZ interactions, and undergoes P(i)-induced internalization. |
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At the apical membrane of renal proximal tubules (PT), the type IIa NaP(i) cotransporter (NaP(i)-IIa) binds specific PDZ domain proteins. Shank2E is a spliceoform of a family of PDZ proteins that is concentrated at the apical domain of liver and pancreatic epithelial cell types and is expressed in kidney. In the present study, immunoblotting of enriched plasma membrane fractions and immunohistology found Shank2E concentrated at the brush border membrane of rat PT cells. Confocal localization of Flag-Shank2E and enhanced green fluorescent protein-NaP(i)-IIa in cotransfected OK cells showed these proteins colocalized in the apical microvilli of this PT cell model. Shank2E co-immunoprecipitated with NaP(i)-IIa from rat renal cortex tissue and HA-NaP(i)-IIa coprecipitated with Flag-Shank2E in cotransfected human embryonic kidney HEK cells. Domain analysis showed that the PDZ domain of Shank2E specifically bound NaP(i)-IIa and truncation of the COOH-terminal TRL motif from NaP(i)-IIa abolished this binding, and Far Western blotting showed that the Shank2E- NaP(i)-IIa interaction occurred directly between the two proteins. NaP(i)-IIa activity is regulated by moderating its abundance in the apical membrane. High-P(i) conditions induce NaP(i)-IIa internalization and degradation. In both rat kidney PT cells and OK cells, shifting to high-P(i) conditions induced an acute internal redistribution of Shank2E and, in OK cells, a significant degree of degradation. In sum, Shank2E is concentrated in the apical domain of renal PT cells, specifically binds NaP(i)-IIa via PDZ interactions, and undergoes P(i)-induced internalization.</description><identifier>ISSN: 0363-6143</identifier><identifier>PMID: 15917299</identifier><language>eng</language><publisher>United States</publisher><subject>Adaptor Proteins, Signal Transducing - metabolism ; Animals ; Base Sequence ; Cell Membrane - metabolism ; Cells, Cultured ; Gene Expression ; Kidney Tubules, Proximal - metabolism ; Male ; Molecular Sequence Data ; Phosphates ; Protein Binding ; Protein Isoforms ; Protein Structure, Tertiary ; Rats ; Rats, Sprague-Dawley ; Sodium-Phosphate Cotransporter Proteins ; Sodium-Phosphate Cotransporter Proteins, Type IIa ; Symporters - metabolism</subject><ispartof>American Journal of Physiology: Cell Physiology, 2005-10, Vol.289 (4), p.C1042-C1051</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15917299$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>McWilliams, Ryan R</creatorcontrib><creatorcontrib>Breusegem, Sophia Y</creatorcontrib><creatorcontrib>Brodsky, Kelley F</creatorcontrib><creatorcontrib>Kim, Eunjoon</creatorcontrib><creatorcontrib>Levi, Moshe</creatorcontrib><creatorcontrib>Doctor, R Brian</creatorcontrib><title>Shank2E binds NaP(i) cotransporter at the apical membrane of proximal tubule cells</title><title>American Journal of Physiology: Cell Physiology</title><addtitle>Am J Physiol Cell Physiol</addtitle><description>Proteins expressing postsynaptic density (PSD)-95/Drosophila disk large (Dlg)/zonula occludens-1 (ZO-1) (PDZ) domains are commonly involved in moderating receptor, channel, and transporter activities at the plasma membrane in a variety of cell types. At the apical membrane of renal proximal tubules (PT), the type IIa NaP(i) cotransporter (NaP(i)-IIa) binds specific PDZ domain proteins. Shank2E is a spliceoform of a family of PDZ proteins that is concentrated at the apical domain of liver and pancreatic epithelial cell types and is expressed in kidney. In the present study, immunoblotting of enriched plasma membrane fractions and immunohistology found Shank2E concentrated at the brush border membrane of rat PT cells. Confocal localization of Flag-Shank2E and enhanced green fluorescent protein-NaP(i)-IIa in cotransfected OK cells showed these proteins colocalized in the apical microvilli of this PT cell model. Shank2E co-immunoprecipitated with NaP(i)-IIa from rat renal cortex tissue and HA-NaP(i)-IIa coprecipitated with Flag-Shank2E in cotransfected human embryonic kidney HEK cells. Domain analysis showed that the PDZ domain of Shank2E specifically bound NaP(i)-IIa and truncation of the COOH-terminal TRL motif from NaP(i)-IIa abolished this binding, and Far Western blotting showed that the Shank2E- NaP(i)-IIa interaction occurred directly between the two proteins. NaP(i)-IIa activity is regulated by moderating its abundance in the apical membrane. High-P(i) conditions induce NaP(i)-IIa internalization and degradation. In both rat kidney PT cells and OK cells, shifting to high-P(i) conditions induced an acute internal redistribution of Shank2E and, in OK cells, a significant degree of degradation. In sum, Shank2E is concentrated in the apical domain of renal PT cells, specifically binds NaP(i)-IIa via PDZ interactions, and undergoes P(i)-induced internalization.</description><subject>Adaptor Proteins, Signal Transducing - metabolism</subject><subject>Animals</subject><subject>Base Sequence</subject><subject>Cell Membrane - metabolism</subject><subject>Cells, Cultured</subject><subject>Gene Expression</subject><subject>Kidney Tubules, Proximal - metabolism</subject><subject>Male</subject><subject>Molecular Sequence Data</subject><subject>Phosphates</subject><subject>Protein Binding</subject><subject>Protein Isoforms</subject><subject>Protein Structure, Tertiary</subject><subject>Rats</subject><subject>Rats, Sprague-Dawley</subject><subject>Sodium-Phosphate Cotransporter Proteins</subject><subject>Sodium-Phosphate Cotransporter Proteins, Type IIa</subject><subject>Symporters - metabolism</subject><issn>0363-6143</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNo1kEtLw0AUhWeh2Fr9CzIr0UUgk3kkdymlWqGoaPfhzotG8zIzAf33jlhXF875OHzcE7LMueKZYoIvyHkI73mei0LBGVkwCawsAJbk9e2A_UexobrpbaBP-HLT3FIzxAn7MA5TdBPFSOPBURwbgy3tXKdT6ejg6TgNX02XwjjruXXUuLYNF-TUYxvc5fGuyP5-s19vs93zw-P6bpeNUkCGTHrDAETJreCVEl6AKkB7pxFQSg6CSaORWw_SWsM06krnBbAKEyf5ilz_zSaJz9mFWHdN-BVIbsMcalXJslQ5T-DVEZx152w9Tkl5-q7_n8B_AGrpVxs</recordid><startdate>200510</startdate><enddate>200510</enddate><creator>McWilliams, Ryan R</creator><creator>Breusegem, Sophia Y</creator><creator>Brodsky, Kelley F</creator><creator>Kim, Eunjoon</creator><creator>Levi, Moshe</creator><creator>Doctor, R Brian</creator><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7X8</scope></search><sort><creationdate>200510</creationdate><title>Shank2E binds NaP(i) cotransporter at the apical membrane of proximal tubule cells</title><author>McWilliams, Ryan R ; Breusegem, Sophia Y ; Brodsky, Kelley F ; Kim, Eunjoon ; Levi, Moshe ; Doctor, R Brian</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p549-a15fc199473d43864f49629bfeba9a5539415cba3df95ddc1bab8b02918a62953</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Adaptor Proteins, Signal Transducing - metabolism</topic><topic>Animals</topic><topic>Base Sequence</topic><topic>Cell Membrane - metabolism</topic><topic>Cells, Cultured</topic><topic>Gene Expression</topic><topic>Kidney Tubules, Proximal - metabolism</topic><topic>Male</topic><topic>Molecular Sequence Data</topic><topic>Phosphates</topic><topic>Protein Binding</topic><topic>Protein Isoforms</topic><topic>Protein Structure, Tertiary</topic><topic>Rats</topic><topic>Rats, Sprague-Dawley</topic><topic>Sodium-Phosphate Cotransporter Proteins</topic><topic>Sodium-Phosphate Cotransporter Proteins, Type IIa</topic><topic>Symporters - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>McWilliams, Ryan R</creatorcontrib><creatorcontrib>Breusegem, Sophia Y</creatorcontrib><creatorcontrib>Brodsky, Kelley F</creatorcontrib><creatorcontrib>Kim, Eunjoon</creatorcontrib><creatorcontrib>Levi, Moshe</creatorcontrib><creatorcontrib>Doctor, R Brian</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>MEDLINE - Academic</collection><jtitle>American Journal of Physiology: Cell Physiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>McWilliams, Ryan R</au><au>Breusegem, Sophia Y</au><au>Brodsky, Kelley F</au><au>Kim, Eunjoon</au><au>Levi, Moshe</au><au>Doctor, R Brian</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Shank2E binds NaP(i) cotransporter at the apical membrane of proximal tubule cells</atitle><jtitle>American Journal of Physiology: Cell Physiology</jtitle><addtitle>Am J Physiol Cell Physiol</addtitle><date>2005-10</date><risdate>2005</risdate><volume>289</volume><issue>4</issue><spage>C1042</spage><epage>C1051</epage><pages>C1042-C1051</pages><issn>0363-6143</issn><abstract>Proteins expressing postsynaptic density (PSD)-95/Drosophila disk large (Dlg)/zonula occludens-1 (ZO-1) (PDZ) domains are commonly involved in moderating receptor, channel, and transporter activities at the plasma membrane in a variety of cell types. At the apical membrane of renal proximal tubules (PT), the type IIa NaP(i) cotransporter (NaP(i)-IIa) binds specific PDZ domain proteins. Shank2E is a spliceoform of a family of PDZ proteins that is concentrated at the apical domain of liver and pancreatic epithelial cell types and is expressed in kidney. In the present study, immunoblotting of enriched plasma membrane fractions and immunohistology found Shank2E concentrated at the brush border membrane of rat PT cells. Confocal localization of Flag-Shank2E and enhanced green fluorescent protein-NaP(i)-IIa in cotransfected OK cells showed these proteins colocalized in the apical microvilli of this PT cell model. Shank2E co-immunoprecipitated with NaP(i)-IIa from rat renal cortex tissue and HA-NaP(i)-IIa coprecipitated with Flag-Shank2E in cotransfected human embryonic kidney HEK cells. Domain analysis showed that the PDZ domain of Shank2E specifically bound NaP(i)-IIa and truncation of the COOH-terminal TRL motif from NaP(i)-IIa abolished this binding, and Far Western blotting showed that the Shank2E- NaP(i)-IIa interaction occurred directly between the two proteins. NaP(i)-IIa activity is regulated by moderating its abundance in the apical membrane. High-P(i) conditions induce NaP(i)-IIa internalization and degradation. In both rat kidney PT cells and OK cells, shifting to high-P(i) conditions induced an acute internal redistribution of Shank2E and, in OK cells, a significant degree of degradation. In sum, Shank2E is concentrated in the apical domain of renal PT cells, specifically binds NaP(i)-IIa via PDZ interactions, and undergoes P(i)-induced internalization.</abstract><cop>United States</cop><pmid>15917299</pmid></addata></record> |
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subjects | Adaptor Proteins, Signal Transducing - metabolism Animals Base Sequence Cell Membrane - metabolism Cells, Cultured Gene Expression Kidney Tubules, Proximal - metabolism Male Molecular Sequence Data Phosphates Protein Binding Protein Isoforms Protein Structure, Tertiary Rats Rats, Sprague-Dawley Sodium-Phosphate Cotransporter Proteins Sodium-Phosphate Cotransporter Proteins, Type IIa Symporters - metabolism |
title | Shank2E binds NaP(i) cotransporter at the apical membrane of proximal tubule cells |
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