Ubiquitylation of leptin receptor OB-Ra regulates its clathrin-mediated endocytosis

EMBO J. 2006 Mar 8;25(5):932-42. doi: 10.1038/sj.emboj.7600989. Epub 2006 Feb 16.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Membrane
  • Clathrin / metabolism*
  • Endocytosis*
  • Endosomes / metabolism
  • Enzyme Inhibitors / pharmacology
  • HeLa Cells
  • Humans
  • Mutation / genetics
  • Proteasome Inhibitors
  • Protein Isoforms
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / metabolism*
  • Receptors, Leptin
  • Receptors, Transferrin / metabolism
  • Ubiquitin / metabolism*

Substances

  • Clathrin
  • Enzyme Inhibitors
  • Proteasome Inhibitors
  • Protein Isoforms
  • Receptors, Cell Surface
  • Receptors, Leptin
  • Receptors, Transferrin
  • Ubiquitin
  • leptin receptor, human