Mutations in the gene encoding the kidney anion exchanger 1 (kAE1) can lead to distal renal tubular acidosis (dRTA). dRTA mutations reported within the carboxyl (C)-terminal tail of kAE1 result in apical mis-targeting of the exchanger in polarized renal epithelial cells. As kAE1 physically interacts with the μ subunit of epithelial adaptor protein 1 B (AP-1B), we investigated the role of heterologously expressed μ1B subunit of the AP-1B complex for kAE1 retention to the basolateral membrane in polarized porcine LLC-PK1 renal epithelial cells that are devoid of endogenous AP-1B. We confirmed the interaction and close proximity between kAE1 and μ1B using immunoprecipitation and proximity ligation assay, respectively. Expressing the human μ1B subunit in these cells decreased significantly the amount of cell surface kAE1 at the steady state, but had no significant effect on kAE1 recycling and endocytosis. We show that (i) heterologous expression of μ1B displaces the physical interaction of endogenous GAPDH with kAE1 WT supporting that both AP-1B and GAPDH proteins bind to an overlapping site on kAE1 and (ii) phosphorylation of tyrosine 904 within the potential YDEV interaction motif does not alter the kAE1/AP-1B interaction. We conclude that μ1B subunit is not involved in recycling of kAE1.

编码肾脏阴离子交换剂1（kAE1）的基因中的突变可导致远端肾小管性酸中毒（dRTA）。在kAE1的羧基（C）末端尾巴中报道的dRTA突变导致极化的肾上皮细胞中交换子的顶端错误靶向。由于kAE1与上皮衔接蛋白1 B（AP-1B）的μ亚基发生物理相互作用，我们研究了异源表达的AP-1B复合物的μ1B亚基对于kAE1保留在极化猪LLC-PK1肾上皮的基底外侧膜中的作用。不含内源性AP-1B的细胞。我们分别使用免疫沉淀法和邻近结扎法确认了kAE1和μ1B之间的相互作用和紧密接近。在稳定状态下，在这些细胞中表达人μ1B亚基会显着降低细胞表面kAE1的数量，但对kAE1的回收和内吞作用无明显影响。我们显示（i）μ1B的异源表达取代了内源性GAPDH与kAE1 WT的物理相互作用，从而支持AP-1B和GAPDH蛋白都结合到kAE1上的重叠位点，以及（ii）潜在YDEV相互作用基序中酪氨酸904的磷酸化不会改变kAE1 / AP-1B的相互作用。我们得出的结论是，μ1B亚基不参与kAE1的回收。