title: Annexin A2 mediates apical trafficking of renal Na⁺-K⁺-2Cl⁻ cotransporter.
abstract: The furosemide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) is responsible for urine concentration and helps maintain systemic salt homeostasis. Its activity depends on trafficking to, and insertion into, the apical membrane, as well as on phosphorylation of conserved N-terminal serine and threonine residues. Vasopressin (AVP) signaling via PKA and other kinases activates NKCC2. Association of NKCC2 with lipid rafts facilitates its AVP-induced apical translocation and activation at the surface. Lipid raft microdomains typically serve as platforms for membrane proteins to facilitate their interactions with other proteins, but little is known about partners that interact with NKCC2. Yeast two-hybrid screening identified an interaction between NKCC2 and the cytosolic protein, annexin A2 (AnxA2). Annexins mediate lipid raft-dependent trafficking of transmembrane proteins, including the AVP-regulated water channel, aquaporin 2. Here, we demonstrate that AnxA2, which binds to phospholipids in a Ca(2+)-dependent manner and may organize microdomains, is codistributed with NKCC2 to promote its apical translocation in response to AVP stimulation and low chloride hypotonic stress. NKCC2 and AnxA2 interact in a phosphorylation-dependent manner. Phosphomimetic AnxA2 carrying a mutant phosphoacceptor (AnxA2-Y24D-GFP) enhanced surface expression and raft association of NKCC2 by 5-fold upon low chloride hypotonic stimulation, whereas AnxA2-Y24A-GFP and PKC-dependent AnxA2-S26D-GFP did not. As the AnxA2 effect involved only nonphosphorylated NKCC2, it appears to affect NKCC2 trafficking. Overexpression or knockdown experiments further supported the role of AnxA2 in the apical translocation and surface expression of NKCC2. In summary, this study identifies AnxA2 as a lipid raft-associated trafficking factor for NKCC2 and provides mechanistic insight into the regulation of this essential cotransporter.
authors: From the Department of Anatomy, Charité-Universitätsmedizin Berlin, 10115 Berlin.
authors: Christin
authors: C
authors: Dathe
authors: Anna-Lena
authors: AL
authors: Daigeler
authors: Wenke
authors: W
authors: Seifert
authors: Vera
authors: V
authors: Jankowski
authors: Ralf
authors: R
authors: Mrowka
authors: Ronny
authors: R
authors: Kalis
authors: Erich
authors: E
authors: Wanker
authors: Kerim
authors: K
authors: Mutig
authors: Sebastian
authors: S
authors: Bachmann
authors: Alexander
authors: A
authors: Paliege
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D017306:Annexin A2 / Q000235:genetics / Q000378:metabolism*; D050034:Antidiuretic Agents / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008253:Macaca mulatta; D008297:Male; D021962:Membrane Microdomains / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D064466:Solute Carrier Family 12, Member 1 / Q000235:genetics / Q000378:metabolism*; D014667:Vasopressins / Q000494:pharmacology
chemical_list: D017306:Annexin A2; D050034:Antidiuretic Agents; C578716:Slc12a1 protein, rat; D064466:Solute Carrier Family 12, Member 1; D014667:Vasopressins
keywords: Annexin; Chloride Channels; Kidney; Phosphorylation; Trafficking