Ubiquitylation of leptin receptor OB-Ra regulates its clathrin-mediated endocytosis.
Leptin receptors are constitutively endocytosed in a ligand-independent manner. To study their endocytosis, leptin receptors OB-Ra and OB-Rb were expressed in HeLa cells. Both receptor isoforms were ubiquitylated, internalized by clathrin-mediated endocytosis and transported to Hrs-positive endosomes after their internalization. Proteasome inhibitors inhibited OB-Ra but not OB-Rb internalization from the cell surface. OB-Ra ubiquitylation occurred on lysine residues K877 and K889 in the cytoplasmic tail, the mutation of which abolished OB-Ra internalization. Fusion of an ubiquitin molecule at the C-terminus of an OB-Ra construct defective both in ubiquitylation and endocytosis restored clathrin-dependent endocytosis of the receptor. The internalization of this constitutively mono-ubiquitylated construct was no longer sensitive to proteasome inhibitors, which inhibited OB-Ra endocytosis by blocking its ubiquitylation. Fusion of an ubiquitin molecule to a transferrin receptor deleted from its own endocytosis motif restored clathrin-mediated endocytosis. We propose that mono-ubiquitin conjugates act as internalization motifs for clathrin-dependent endocytosis of leptin receptor OB-Ra.
Centre National de la Recherche Scientifique, Unité Propre de Recherche 2511, Institut Pasteur de Lille, Lille Cedex, France.
Sandrine
S
Belouzard
Yves
Y
Rouillé
D002462:Cell Membrane; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D009154:Mutation / Q000235:genetics; D061988:Proteasome Inhibitors; D020033:Protein Isoforms; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D054411:Receptors, Leptin; D011990:Receptors, Transferrin / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*
D002966:Clathrin; D004791:Enzyme Inhibitors; C515448:LEPR protein, human; D061988:Proteasome Inhibitors; D020033:Protein Isoforms; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D011990:Receptors, Transferrin; D025801:Ubiquitin