title: Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1.
abstract: Kidney anion exchanger 1 (kAE1) mediates chloride/bicarbonate exchange at the basolateral membrane of kidney alpha-intercalated cells, thereby facilitating bicarbonate reabsorption into the blood. Human kAE1 lacks the N-terminal 65 residues of the erythroid form (AE1, band 3), which are essential for binding of cytoskeletal and cytosolic proteins. Yeast two-hybrid screening identified integrin-linked kinase (ILK), a serine/threonine kinase, and an actin-binding protein as an interacting partner with the N-terminal domain of kAE1. Interaction between kAE1 and ILK was confirmed in co-expression experiments in HEK 293 cells and is mediated by a previously unidentified calponin homology domain in the kAE1 N-terminal region. The calponin homology domain of kAE1 binds the C-terminal catalytic domain of ILK to enhance association of kAE1 with the actin cytoskeleton. Overexpression of ILK increased kAE1 levels at the cell surface as shown by flow cytometry, cell surface biotinylation, and anion transport activity assays. Pulse-chase experiments revealed that ILK associates with kAE1 early in biosynthesis, likely in the endoplasmic reticulum. ILK co-localized with kAE1 at the basolateral membrane of polarized Madin-Darby canine kidney cells and in alpha-intercalated cells of human kidneys. Taken together these results suggest that ILK and kAE1 traffic together from the endoplasmic reticulum to the basolateral membrane. ILK may provide a linkage between kAE1 and the underlying actin cytoskeleton to stabilize kAE1 at the basolateral membrane, resulting in higher levels of cell surface expression.
authors.affiliation: Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Thitima
authors.initials: T
authors.lastname: Keskanokwong
authors.forename: Haley J
authors.initials: HJ
authors.lastname: Shandro
authors.forename: Danielle E
authors.initials: DE
authors.lastname: Johnson
authors.forename: Saranya
authors.initials: S
authors.lastname: Kittanakom
authors.forename: Gonzalo L
authors.initials: GL
authors.lastname: Vilas
authors.forename: Paul
authors.initials: P
authors.lastname: Thorner
authors.forename: Reinhart A F
authors.initials: RA
authors.lastname: Reithmeier
authors.forename: Varaporn
authors.initials: V
authors.lastname: Akkarapatumwong
authors.forename: Pa-thai
authors.initials: PT
authors.lastname: Yenchitsomanus
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Casey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques; D000099200:Scaffold Protein ILK
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D017346:Protein Serine-Threonine Kinases; D000099200:Scaffold Protein ILK