The ARH adaptor protein regulates endocytosis of the ROMK potassium secretory channel in mouse kidney
Liang Fang, … , James B. Wade, Paul A. Welling
Liang Fang, … , James B. Wade, Paul A. Welling
Published October 19, 2009
Citation Information: J Clin Invest. 2009;119(11):3278-3289. https://doi.org/10.1172/JCI37950.
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Research Article Nephrology

The ARH adaptor protein regulates endocytosis of the ROMK potassium secretory channel in mouse kidney

Abstract

Renal outer medullary potassium (ROMK) channels are exquisitely regulated to adjust renal potassium excretion and maintain potassium balance. Clathrin-dependent endocytosis plays a critical role, limiting urinary potassium loss in potassium deficiency. In renal disease, aberrant ROMK endocytosis may contribute to potassium retention and hyperkalemia. Previous work has indicated that ROMK endocytosis is stimulated by with-no-lysine (WNK) kinases, but the endocytotic signal and the internalization machinery have not been defined. Here, we found that ROMK bound directly to the clathrin adaptor molecule autosomal recessive hypercholesterolemia (ARH), and this interaction was mediated by what we believe to be a novel variant of the canonical “NPXY” endocytotic signal, YxNPxFV. ARH recruits ROMK to clathrin-coated pits for constitutive and WNK1-stimuated endocytosis, and ARH knockdown decreased basal rates of ROMK endocytosis, in a heterologous expression system, COS-7 cells. We found that ARH was predominantly expressed in the distal nephron where it coimmunoprecipitated and colocalized with ROMK. In mice, the abundance of kidney ARH protein was modulated by dietary potassium and inversely correlated with changes in ROMK. Furthermore, ARH-knockout mice exhibited an altered ROMK response to potassium intake. These data suggest that ARH marks ROMK for clathrin-dependent endocytosis, in concert with the demands of potassium homeostasis.

Authors

Liang Fang, Rita Garuti, Bo-Young Kim, James B. Wade, Paul A. Welling

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Figure 1

ROMK preferentially interacts with ARH.

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ROMK preferentially interacts with ARH. (A) Illustration of the pull-dow...
(A) Illustration of the pull-down assay. Recombinant His-tagged PTB-CLASPs (ARH, Dab2, Gulp, and Numb) were bound to Ni-NTA beads and tested for interaction with GST fusion proteins of either the ROMK C terminus or the LRP C terminus. H5, His-tag. (B) GST fusions of the WT ROMK1 C terminus (ROMK-C) (amino acids 349–391), LRP C terminus (LRP-C), and His-tagged PTB proteins were purified to homogeneity for the binding assay. Shown are purified proteins from E. coli–expressing indicated GST protein or His-tag CLASP, which had been resolved by SDS-PAGE and visualized by Coomassie Brilliant Blue staining and compared with vector-only–transfected E. coli (E. coli, BL21). (C) Binding assay. After incubation of GST proteins with each of indicated PTB-CLASPs, specifically bound protein (GST alone, GST ROMK C terminus [GST-ROMK-C], and GST LRP C terminus [GST-LRP-C] proteins) was assessed by immunoblot with anti-GST antibodies and compared with input GST (middle row) and HIS-tagged proteins (bottom row). GST-ROMK-C preferentially bound to ARH, while GST-LRP-C bound all the PTB-CLASPs. As observed before (23), GST-LRP-C migrates as doublet due to partial proteolysis. (D and E) ARH interacts with ROMK in an NPXF signal–dependent manner. Binding of ARH with GST, GST-ROMK-C, and a mutant GST-ROMK-C, bearing an alanine replacement of the N375PNF sequence (-NPNF). (E) Quantification of binding. n = 3; *P < 0.001.