Role of CIC-5 in Renal Endocytosis Is Unique among CIC Exchangers and Does Not Require PY-motif-dependent Ubiquitylation

• DOI: 10.1074/jbc.m110.115600
• Publication Date: 2010-03-30T02:24:30Z
• Authors: Gesa Rickheit; Lena Wartosch; Sven Schaffer; Sandra M. Stobrawa; Gaia Novarino; Stefanie Weinert; Thomas J. Jentsch
• Keywords: Male; Mice, Knockout; 0301 basic medicine; Cytoplasm; Ubiquitin; Amino Acid Motifs; WW-domain ; Endocytosis ; Mouse Genetics ; Kidney Metabolism ; Chloride Channels ; Membrane Trafficking ; Anion/Proton Antiporter ; Endosomal Acidification; Models, Biological; Endocytosis; Protein Structure, Tertiary; Low Density Lipoprotein Receptor-Related Protein-2; Mice; 03 medical and health sciences; Kidney Tubules; Gene Expression Regulation; Chloride Channels; Animals; Female

Inactivation of the mainly endosomal 2Cl(-)/H(+)-exchanger ClC-5 severely impairs endocytosis in renal proximal tubules and underlies the human kidney stone disorder Dent's disease. In heterologous expression systems, interaction of the E3 ubiquitin ligases WWP2 and Nedd4-2 with a "PY-motif" in the cytoplasmic C terminus of ClC-5 stimulates its internalization from the plasma membrane and may influence receptor-mediated endocytosis. We asked whether this interaction is relevant in vivo and generated mice in which the PY-motif was destroyed by a point mutation. Unlike ClC-5 knock-out mice, these knock-in mice displayed neither low molecular weight proteinuria nor hyperphosphaturia, and both receptor-mediated and fluid-phase endocytosis were normal. The abundances and localizations of the endocytic receptor megalin and of the Na(+)-coupled phosphate transporter NaPi-2a (Npt2) were not changed, either. To explore whether the discrepancy in results from heterologous expression studies might be due to heteromerization of ClC-5 with ClC-3 or ClC-4 in vivo, we studied knock-in mice additionally deleted for those related transporters. Disruption of neither ClC-3 nor ClC-4 led to proteinuria or impaired proximal tubular endocytosis by itself, nor in combination with the PY-mutant of ClC-5. Endocytosis of cells lacking ClC-5 was not impaired further when ClC-3 or ClC-4 was additionally deleted. We conclude that ClC-5 is unique among CLC proteins in being crucial for proximal tubular endocytosis and that PY-motif-dependent ubiquitylation of ClC-5 is dispensable for this role.