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.
Hislop
James N
JN
Departments of Psychiatry and Cellular and Molecular Pharmacology, University of California, San Francisco, California 94158, USA. james.hislop@ucsf.edu
Henry
Anastasia G
AG
Marchese
Adriano
A
von Zastrow
Mark
M
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