Recycling of The Kidney Anion Exchanger 1 Is Regulated by Adaptor Protein Complex 1B

Abstract

Adaptor protein complexes 1 (AP‐1) regulate polarized trafficking of membrane proteins. Here, we investigated the role of AP‐1B in regulating the kidney anion exchanger 1 (kAE1) intracellular trafficking. kAE1 physically interacts with AP‐1B in Madin‐Darby canine kidney cells. We hypothesized that AP‐1B regulates the recycling of endocytosed kAE1 protein and that kAE1 mutants that cause distal renal tubular acidosis, fail to properly interact with AP‐1B. We studied kAE1 WT and a dRTA mutant, kAE1 R901X, which lacks the last 11 amino acids encompassing the potential canonical binding site for AP‐1B. We confirmed the physical interaction between kAE1 and heterologously expressed μ1B subunit of AP‐1B in LLC‐PK1 renal epithelial cells lacking endogenous AP‐1B. A peptide spot assay identified multiple interaction sites of AP‐1B in the kAE1 protein sequence, including residues within the second and sixth cytosolic loops, and the C‐terminus. μ1B heterologous expression decreased the amount of plasma membrane kAE1. Although kAE1 R901X mutant underwent endocytosis slightly faster than kAE1 WT, its recycling rate was decreased compared to kAE1 WT, and was independent from μ1B expression. We also observed that (i) μ1B expression disrupts the interaction of GAPDH with kAE1 WT consistent with both proteins binding to an overlapping site on kAE1, and (ii) phosphorylation of tyrosine 904 within the carboxyl‐terminal interaction motif does not affect the kAE1/AP‐1B interaction. We propose that the apically mis‐targeted kAE1 R901X mutant fails to recycle back to the basolateral membrane due to its inability to properly interact with AP‐1B.