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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Published in:	Biomolecules (Basel, Switzerland) Switzerland), 2022-04, Vol.12 (5), p.633
Main Authors:	Pati, Roberta, Palazzo, Claudia, Valente, Onofrio, Abbrescia, Pasqua, Messina, Raffaella, Surdo, Nicoletta Concetta, Lefkimmiatis, Konstantinos, Signorelli, Francesco, Nicchia, Grazia Paola, Frigeri, Antonio
Format:	Article
Language:	eng
Subjects:	Animals Antibodies Aquaporin 4 Aquaporin 4 - genetics Aquaporin-4 (AQP4) Aquaporins Astrocytes - metabolism Automation Biopsy Blood-brain barrier Brain Brain - metabolism Channel gating Enzyme-linked immunosorbent assay Experiments Homeostasis Humans Immunofluorescence Isoforms Kinases Localization Mammals Mice Mutagenesis p-AQP4ex Penicillin Peptides Phosphorylation Protein Isoforms - metabolism Proteins Rabbits Rats Serine - metabolism short-term regulation Site-directed mutagenesis translational readthrough
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Summary:	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 and serine , 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 is conserved in human, mouse and rat AQP4ex. The phosphorylation site of Ser 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 ( -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 in the extended sequence exclusive of AQP4ex. Therefore, we anticipate an important physiological role of -AQP4ex in human brain water homeostasis.
ISSN:	2218-273X 2218-273X