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The Readthrough Isoform AQP4ex Is Constitutively Phosphorylated in the Perivascular Astrocyte Endfeet of Human Brain

AQP4ex is a recently discovered isoform of AQP4 generated by a translational readthrough mechanism. It is strongly expressed at the astrocyte perivascular endfeet as a component of the supramolecular membrane complex, commonly called orthogonal array of particles (OAP), together with the canonical i...

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Autores principales: Pati, Roberta, Palazzo, Claudia, Valente, Onofrio, Abbrescia, Pasqua, Messina, Raffaella, Surdo, Nicoletta Concetta, Lefkimmiatis, Konstantinos, Signorelli, Francesco, Nicchia, Grazia Paola, Frigeri, Antonio
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9138620/
https://www.ncbi.nlm.nih.gov/pubmed/35625560
http://dx.doi.org/10.3390/biom12050633
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author Pati, Roberta
Palazzo, Claudia
Valente, Onofrio
Abbrescia, Pasqua
Messina, Raffaella
Surdo, Nicoletta Concetta
Lefkimmiatis, Konstantinos
Signorelli, Francesco
Nicchia, Grazia Paola
Frigeri, Antonio
author_facet Pati, Roberta
Palazzo, Claudia
Valente, Onofrio
Abbrescia, Pasqua
Messina, Raffaella
Surdo, Nicoletta Concetta
Lefkimmiatis, Konstantinos
Signorelli, Francesco
Nicchia, Grazia Paola
Frigeri, Antonio
author_sort Pati, Roberta
collection PubMed
description AQP4ex is a recently discovered isoform of AQP4 generated by a translational readthrough mechanism. It is strongly expressed at the astrocyte perivascular endfeet as a component of the supramolecular membrane complex, commonly called orthogonal array of particles (OAP), together with the canonical isoforms M1 and M23 of AQP4. Previous site-directed mutagenesis experiments suggested the potential role of serine(331) and serine(335), located in the extended peptide of AQP4ex, in water channel activity by phosphorylation. In the present study we evaluated the effective phosphorylation of human AQP4ex. A small scale bioinformatic analysis indicated that only Ser(335) is conserved in human, mouse and rat AQP4ex. The phosphorylation site of Ser(335) was assessed through generation of phospho-specific antibodies in rabbits. Antibody specificity was first evaluated in binding phosphorylated peptide versus its unphosphorylated analog by ELISA, which was further confirmed by site-directed mutagenesis experiments. Western blot and immunofluorescence experiments revealed strong expression of phosphorylated AQP4ex (p-AQP4ex) in human brain and localization at the perivascular astrocyte endfeet in supramolecular assemblies identified by BN/PAGE experiments. All together, these data reveal, for the first time, the existence of a phosphorylated form of AQP4, at Ser(335) in the extended sequence exclusive of AQP4ex. Therefore, we anticipate an important physiological role of p-AQP4ex in human brain water homeostasis.
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spelling pubmed-91386202022-05-28 The Readthrough Isoform AQP4ex Is Constitutively Phosphorylated in the Perivascular Astrocyte Endfeet of Human Brain Pati, Roberta Palazzo, Claudia Valente, Onofrio Abbrescia, Pasqua Messina, Raffaella Surdo, Nicoletta Concetta Lefkimmiatis, Konstantinos Signorelli, Francesco Nicchia, Grazia Paola Frigeri, Antonio Biomolecules Article AQP4ex is a recently discovered isoform of AQP4 generated by a translational readthrough mechanism. It is strongly expressed at the astrocyte perivascular endfeet as a component of the supramolecular membrane complex, commonly called orthogonal array of particles (OAP), together with the canonical isoforms M1 and M23 of AQP4. Previous site-directed mutagenesis experiments suggested the potential role of serine(331) and serine(335), located in the extended peptide of AQP4ex, in water channel activity by phosphorylation. In the present study we evaluated the effective phosphorylation of human AQP4ex. A small scale bioinformatic analysis indicated that only Ser(335) is conserved in human, mouse and rat AQP4ex. The phosphorylation site of Ser(335) was assessed through generation of phospho-specific antibodies in rabbits. Antibody specificity was first evaluated in binding phosphorylated peptide versus its unphosphorylated analog by ELISA, which was further confirmed by site-directed mutagenesis experiments. Western blot and immunofluorescence experiments revealed strong expression of phosphorylated AQP4ex (p-AQP4ex) in human brain and localization at the perivascular astrocyte endfeet in supramolecular assemblies identified by BN/PAGE experiments. All together, these data reveal, for the first time, the existence of a phosphorylated form of AQP4, at Ser(335) in the extended sequence exclusive of AQP4ex. Therefore, we anticipate an important physiological role of p-AQP4ex in human brain water homeostasis. MDPI 2022-04-25 /pmc/articles/PMC9138620/ /pubmed/35625560 http://dx.doi.org/10.3390/biom12050633 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Pati, Roberta
Palazzo, Claudia
Valente, Onofrio
Abbrescia, Pasqua
Messina, Raffaella
Surdo, Nicoletta Concetta
Lefkimmiatis, Konstantinos
Signorelli, Francesco
Nicchia, Grazia Paola
Frigeri, Antonio
The Readthrough Isoform AQP4ex Is Constitutively Phosphorylated in the Perivascular Astrocyte Endfeet of Human Brain
title The Readthrough Isoform AQP4ex Is Constitutively Phosphorylated in the Perivascular Astrocyte Endfeet of Human Brain
title_full The Readthrough Isoform AQP4ex Is Constitutively Phosphorylated in the Perivascular Astrocyte Endfeet of Human Brain
title_fullStr The Readthrough Isoform AQP4ex Is Constitutively Phosphorylated in the Perivascular Astrocyte Endfeet of Human Brain
title_full_unstemmed The Readthrough Isoform AQP4ex Is Constitutively Phosphorylated in the Perivascular Astrocyte Endfeet of Human Brain
title_short The Readthrough Isoform AQP4ex Is Constitutively Phosphorylated in the Perivascular Astrocyte Endfeet of Human Brain
title_sort readthrough isoform aqp4ex is constitutively phosphorylated in the perivascular astrocyte endfeet of human brain
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9138620/
https://www.ncbi.nlm.nih.gov/pubmed/35625560
http://dx.doi.org/10.3390/biom12050633
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