title: Evidence of a structural and functional ammonium transporter RhBG·anion exchanger 1·ankyrin-G complex in kidney epithelial cells.
abstract: The renal ammonium transporter RhBG and anion exchanger 1 kAE1 colocalize in the basolateral domain of α-intercalated cells in the distal nephron. Although we have previously shown that RhBG is linked to the spectrin-based skeleton through ankyrin-G and that its NH3 transport activity is dependent on this association, there is no evidence for an interaction of kAE1 with this adaptor protein. We report here that the kAE1 cytoplasmic N terminus actually binds to ankyrin-G, both in yeast two-hybrid analysis and by coimmunoprecipitation in situ in HEK293 cells expressing recombinant kAE1. A site-directed mutagenesis study allowed the identification of three dispersed regions on kAE1 molecule linking the third and fourth repeat domains of ankyrin-G. One secondary docking site corresponds to a major interacting loop of the erythroid anion exchanger 1 (eAE1) with ankyrin-R, whereas the main binding region of kAE1 does not encompass any eAE1 determinant. Stopped flow spectrofluorometry analysis of recombinant HEK293 cells revealed that the Cl(-)/HCO3 (-) exchange activity of a kAE1 protein mutated on the ankyrin-G binding site was abolished. This disruption impaired plasma membrane expression of kAE1 leading to total retention on cytoplasmic structures in polarized epithelial Madin-Darby canine kidney cell transfectants. kAE1 also directly interacts with RhBG without affecting its surface expression and NH3 transport function. This is the first description of a structural and functional RhBG·kAE1·ankyrin-G complex at the plasma membrane of kidney epithelial cells, comparable with the well known Rh·eAE1·ankyrin-R complex in the red blood cell membrane. This renal complex could participate in the regulation of acid-base homeostasis.
authors.affiliation: From INSERM U1134, 75739 Paris, France, the Université Paris Diderot, Sorbonne Paris Cité, UMR_S1134, 75739 Paris, France, the Institut National de la Transfusion Sanguine, 75739 Paris, France, and the Laboratoire d'Excellence GR-Ex, 75238 Paris, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Genetet
authors.affiliation: From INSERM U1134, 75739 Paris, France, the Université Paris Diderot, Sorbonne Paris Cité, UMR_S1134, 75739 Paris, France, the Institut National de la Transfusion Sanguine, 75739 Paris, France, and the Laboratoire d'Excellence GR-Ex, 75238 Paris, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Ripoche
authors.affiliation: From INSERM U1134, 75739 Paris, France, the Université Paris Diderot, Sorbonne Paris Cité, UMR_S1134, 75739 Paris, France, the Institut National de la Transfusion Sanguine, 75739 Paris, France, and the Laboratoire d'Excellence GR-Ex, 75238 Paris, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Le Van Kim
authors.affiliation: From INSERM U1134, 75739 Paris, France, the Université Paris Diderot, Sorbonne Paris Cité, UMR_S1134, 75739 Paris, France, the Institut National de la Transfusion Sanguine, 75739 Paris, France, and the Laboratoire d'Excellence GR-Ex, 75238 Paris, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Colin
authors.affiliation: From INSERM U1134, 75739 Paris, France, the Université Paris Diderot, Sorbonne Paris Cité, UMR_S1134, 75739 Paris, France, the Institut National de la Transfusion Sanguine, 75739 Paris, France, and the Laboratoire d'Excellence GR-Ex, 75238 Paris, France claude.lopez@inserm.fr.
authors.forename: Claude
authors.initials: C
authors.lastname: Lopez
mesh_terms: D064751:Ammonium Compounds / Q000378:metabolism*; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D017487:Ankyrins / Q000032:analysis / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism*; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007668:Kidney / Q000166:cytology*; D026901:Membrane Transport Proteins / Q000032:analysis / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D025941:Protein Interaction Mapping; D060066:Protein Interaction Maps
chemical_list: C490613:ANK3 protein, human; D064751:Ammonium Compounds; D001457:Anion Exchange Protein 1, Erythrocyte; D017487:Ankyrins; D006023:Glycoproteins; D026901:Membrane Transport Proteins; C422995:RHBG protein, human
keywords: AE1; Ankryin; Epithelial Cell; Kidney; Membrane Transport; Protein Complex; RhBG