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Multiple phosphorylation sites are present at the C-terminal of AQP2 including S256 (serine at 256 residue), S261, S264 and S\/T269, which are regulated by vasopressin (VP) to modulate AQP2 trafficking. As the dynamics of these phosphorylations have been studied mostly in rodents, little is known about the phosphorylation of human AQP2 which has unique T269 in the place of S269 of rodent AQP2. Because AQP2 is excreted in urinary exosomes, the phosphoprotein profile of human AQP2 can be easily examined through urinary exosomes without any intervention.<\/jats:p>\n<\/jats:sec><jats:sec>\n<jats:title>Methods<\/jats:title>\n<jats:p>Human urinary exosomes digested with trypsin or glutamyl endopeptidase (Glu-C) were examined by the liquid chromatography coupled with tandem mass spectrometry (LC\u2013MS\/MS) phosphoproteomic analysis.<\/jats:p>\n<\/jats:sec><jats:sec>\n<jats:title>Results<\/jats:title>\n<jats:p>The most dominant phosphorylated AQP2 peptide identified was S256 phosphorylated form (pS256), followed by pS261 with less pS264 and far less pT269, which was confirmed by the western blot analyses using phosphorylated AQP2-specific antibodies. In a patient lacking circulating VP, administration of a VP analogue showed a transient increase (peak at 30\u201360\u00a0min) in excretion of exosomes with pS261 AQP2.<\/jats:p>\n<\/jats:sec><jats:sec>\n<jats:title>Conclusion<\/jats:title>\n<jats:p>These data suggest that all phosphorylation sites of human AQP2 including T269 are phosphorylated and phosphorylations at S256 and S261 may play a dominant role in the urinary exosomal excretion of AQP2.<\/jats:p>\n<\/jats:sec>","DOI":"10.1007\/s10157-020-01899-4","type":"journal-article","created":{"date-parts":[[2020,6,11]],"date-time":"2020-06-11T05:02:41Z","timestamp":1591851761000},"page":"762-769","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Phosphorylation profile of human AQP2 in urinary exosomes by LC\u2013MS\/MS phosphoproteomic analysis"],"prefix":"10.1007","volume":"24","author":[{"given":"Masaki","family":"Sakai","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keiko","family":"Yamamoto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroaki","family":"Mizumura","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomoki","family":"Matsumoto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yasuko","family":"Tanaka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yumi","family":"Noda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kenichi","family":"Ishibashi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tadashi","family":"Yamamoto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sei","family":"Sasaki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2020,6,11]]},"reference":[{"key":"1899_CR1","doi-asserted-by":"publisher","first-page":"549","DOI":"10.1038\/361549a0","volume":"361","author":"K Fushimi","year":"1993","unstructured":"Fushimi K, Uchida S, Hara Y, Hirata Y, Marumo F, Sasaki S. 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Also, the CDI patient gave written informed consent and the protocol was approved by the Institutional Ethical Committee of Nitobe Memorial Nakano General Hospital (approved number 16-045). All procedures performed in animals were approved by the Meiji Pharmaceutical University Committee for Ethics of Experimentation and Animal Care (approved number 2005).","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Human and animal rights"}},{"value":"Informed consent was obtained from all individual participants included in the study.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Informed consent"}}]}}