Role of ClC-5 in renal endocytosis is unique among ClC exchangers and does not require PY-motif-dependent ubiquitylation.
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.
Rickheit
Gesa
G
Leibniz-Institut für Molekulare Pharmakologie and Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Strasse 10, D-13125 Berlin, Germany.
Wartosch
Lena
L
Schaffer
Sven
S
Stobrawa
Sandra M
SM
Novarino
Gaia
G
Weinert
Stefanie
S
Jentsch
Thomas J
TJ
D020816:Amino Acid Motifs; D000818:Animals; D018118:Chloride Channels / Q000737:chemistry* / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004705:Endocytosis; D005260:Female; D005786:Gene Expression Regulation; D007684:Kidney Tubules / Q000378:metabolism; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000737:chemistry; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D025801:Ubiquitin / Q000737:chemistry*
C471345:CLC5 protein, mouse; D018118:Chloride Channels; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D025801:Ubiquitin