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Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization
The sorting signals that direct proteins to the apical surface of polarized epithelial cells are complex and can include posttranslational modifications, such as N- and O-linked glycosylation. Efficient apical sorting of the neurotrophin receptor p75 is dependent on its O-glycosylated membrane proxi...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Cell Biology
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3681702/ https://www.ncbi.nlm.nih.gov/pubmed/23637462 http://dx.doi.org/10.1091/mbc.E13-02-0078 |
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author | Youker, Robert T. Bruns, Jennifer R. Costa, Simone A. Rbaibi, Youssef Lanni, Frederick Kashlan, Ossama B. Teng, Haibing Weisz, Ora A. |
author_facet | Youker, Robert T. Bruns, Jennifer R. Costa, Simone A. Rbaibi, Youssef Lanni, Frederick Kashlan, Ossama B. Teng, Haibing Weisz, Ora A. |
author_sort | Youker, Robert T. |
collection | PubMed |
description | The sorting signals that direct proteins to the apical surface of polarized epithelial cells are complex and can include posttranslational modifications, such as N- and O-linked glycosylation. Efficient apical sorting of the neurotrophin receptor p75 is dependent on its O-glycosylated membrane proximal stalk, but how this domain mediates targeting is unknown. Protein oligomerization or clustering has been suggested as a common step in the segregation of all apical proteins. Like many apical proteins, p75 forms dimers, and we hypothesized that formation of higher-order clusters mediated by p75 dimerization and interactions of the stalk facilitate its apical sorting. Using fluorescence fluctuation techniques (photon-counting histogram and number and brightness analyses) to study p75 oligomerization status in vivo, we found that wild-type p75–green fluorescent protein forms clusters in the trans-Golgi network (TGN) but not at the plasma membrane. Disruption of either the dimerization motif or the stalk domain impaired both clustering and polarized delivery. Manipulation of O-glycan processing or depletion of multiple galectins expressed in Madin-Darby canine kidney cells had no effect on p75 sorting, suggesting that the stalk domain functions as a structural prop to position other determinants in the lumenal domain of p75 for oligomerization. Additionally, a p75 mutant with intact dimerization and stalk motifs but with a dominant basolateral sorting determinant (Δ250 mutant) did not form oligomers, consistent with a requirement for clustering in apical sorting. Artificially enhancing dimerization restored clustering to the Δ250 mutant but was insufficient to reroute this mutant to the apical surface. Together these studies demonstrate that clustering in the TGN is required for normal biosynthetic apical sorting of p75 but is not by itself sufficient to reroute a protein to the apical surface in the presence of a strong basolateral sorting determinant. Our studies shed new light on the hierarchy of polarized sorting signals and on the mechanisms by which newly synthesized proteins are segregated in the TGN for eventual apical delivery. |
format | Online Article Text |
id | pubmed-3681702 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-36817022013-08-30 Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization Youker, Robert T. Bruns, Jennifer R. Costa, Simone A. Rbaibi, Youssef Lanni, Frederick Kashlan, Ossama B. Teng, Haibing Weisz, Ora A. Mol Biol Cell Articles The sorting signals that direct proteins to the apical surface of polarized epithelial cells are complex and can include posttranslational modifications, such as N- and O-linked glycosylation. Efficient apical sorting of the neurotrophin receptor p75 is dependent on its O-glycosylated membrane proximal stalk, but how this domain mediates targeting is unknown. Protein oligomerization or clustering has been suggested as a common step in the segregation of all apical proteins. Like many apical proteins, p75 forms dimers, and we hypothesized that formation of higher-order clusters mediated by p75 dimerization and interactions of the stalk facilitate its apical sorting. Using fluorescence fluctuation techniques (photon-counting histogram and number and brightness analyses) to study p75 oligomerization status in vivo, we found that wild-type p75–green fluorescent protein forms clusters in the trans-Golgi network (TGN) but not at the plasma membrane. Disruption of either the dimerization motif or the stalk domain impaired both clustering and polarized delivery. Manipulation of O-glycan processing or depletion of multiple galectins expressed in Madin-Darby canine kidney cells had no effect on p75 sorting, suggesting that the stalk domain functions as a structural prop to position other determinants in the lumenal domain of p75 for oligomerization. Additionally, a p75 mutant with intact dimerization and stalk motifs but with a dominant basolateral sorting determinant (Δ250 mutant) did not form oligomers, consistent with a requirement for clustering in apical sorting. Artificially enhancing dimerization restored clustering to the Δ250 mutant but was insufficient to reroute this mutant to the apical surface. Together these studies demonstrate that clustering in the TGN is required for normal biosynthetic apical sorting of p75 but is not by itself sufficient to reroute a protein to the apical surface in the presence of a strong basolateral sorting determinant. Our studies shed new light on the hierarchy of polarized sorting signals and on the mechanisms by which newly synthesized proteins are segregated in the TGN for eventual apical delivery. The American Society for Cell Biology 2013-06-15 /pmc/articles/PMC3681702/ /pubmed/23637462 http://dx.doi.org/10.1091/mbc.E13-02-0078 Text en © 2013 Youker et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology. |
spellingShingle | Articles Youker, Robert T. Bruns, Jennifer R. Costa, Simone A. Rbaibi, Youssef Lanni, Frederick Kashlan, Ossama B. Teng, Haibing Weisz, Ora A. Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization |
title | Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization |
title_full | Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization |
title_fullStr | Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization |
title_full_unstemmed | Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization |
title_short | Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization |
title_sort | multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3681702/ https://www.ncbi.nlm.nih.gov/pubmed/23637462 http://dx.doi.org/10.1091/mbc.E13-02-0078 |
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