Ubiquitination regulates proteolytic processing of G protein-coupled receptors after their sorting to lysosomes.
Ubiquitination is essential for the endocytic sorting of various G protein-coupled receptors to lysosomes. Here we identify a distinct function of this covalent modification in controlling the later proteolytic processing of receptors. Mutation of all cytoplasmic lysine residues in the murine delta-opioid receptor blocked receptor ubiquitination without preventing ligand-induced endocytosis of receptors or their subsequent delivery to lysosomes, as verified by proteolysis of extramembrane epitope tags and down-regulation of radioligand binding to the transmembrane helices. Surprisingly, a functional screen revealed that the E3 ubiquitin ligase AIP4 specifically controls down-regulation of wild type receptors measured by radioligand binding without detectably affecting receptor delivery to lysosomes defined both immunochemically and biochemically. This specific AIP4-dependent regulation required direct ubiquitination of receptors and was also regulated by two deubiquitinating enzymes, AMSH and UBPY, which localized to late endosome/lysosome membranes containing internalized delta-opioid receptor. These results identify a distinct function of AIP4-dependent ubiquitination in controlling the later proteolytic processing of G protein-coupled receptors, without detectably affecting their endocytic sorting to lysosomes. We propose that ubiquitination or ubiquitination/deubiquitination cycling specifically regulates later proteolytic processing events required for destruction of the receptor's hydrophobic core.
Departments of Psychiatry and Cellular and Molecular Pharmacology, University of California, San Francisco, California 94158, USA. james.hislop@ucsf.edu
James N
JN
Hislop
Anastasia G
AG
Henry
Adriano
A
Marchese
Mark
M
von Zastrow
D001667:Binding, Competitive; D019921:Biotinylation; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008247:Lysosomes / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination
D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D044767:Ubiquitin-Protein Ligases