Abstract
The δ-opioid receptor (DOR) undergoes ligand-induced downregulation by endosomal sorting complex required for transport (ESCRT)-dependent endocytic trafficking to lysosomes. In contrast to a number of other signaling receptors, the DOR can downregulate effectively when its ubiquitination is prevented. We explored the membrane trafficking basis of this behavior. First, we show that internalized DORs traverse the canonical multivesicular body (MVB) pathway and localize to intralumenal vesicles (ILVs). Second, we show that DOR ubiquitination stimulates, but is not essential for, receptor transfer to ILVs and proteolysis of the receptor endodomain. Third, we show that receptor ubiquitination plays no detectable role in the early sorting of internalized DORs out of the recycling pathway. Finally, we show that DORs undergo extensive proteolytic fragmentation in the ectodomain, even when receptor ubiquitination is prevented or ILV formation itself is blocked. Together, these results are sufficient to explain why DORs downregulate effectively in the absence of ubiquitination, and they place a discrete molecular sorting operation in the MVB pathway effectively upstream of the ESCRT. More generally, these findings support the hypothesis that mammalian cells can control the cytoplasmic accessibility of internalized signaling receptors independently from their ultimate trafficking fate.
| Original language | English |
|---|---|
| Pages (from-to) | 170-184 |
| Number of pages | 15 |
| Journal | Traffic |
| Volume | 12 |
| Issue number | 2 |
| Early online date | 22 Dec 2010 |
| DOIs | |
| Publication status | Published - Feb 2011 |
Funding
This work was supported by grants from the NIH (DA010711 and DA012864 to M. v. Z.) and the UK Medical Research Council to M. M. and I. J. W. A. G. H. is a recipient of a predoctoral fellowship from the U.S. National Science Foundation. The authors gratefully acknowledge Kurt Thorn and the Nikon Imaging Center at UCSF for useful advice, and for providing access to fluorescence imaging instrumentation. We thank Adriano Marchese and Harald Stenmark for valuable discussion and sharing essential reagents, Scott Emr, Manoj Pathenveedu, Michael Tanowitz and Paul Temkin for critical comments and suggestions and Aaron Marley, Laurant Coscoy, Roger Tsien and Marino Zerial for generously providing constructs used in this study.
Keywords
- down-regulation
- multivesicular endosomes
- mediates ubiquitination
- binding proteins
- ligase AIP4
- protein-coupled receptor
- ubiquitin
- molecular-mechanisms
- GPCR
- sorting proteins
- membrane
- microscopy
- recycling pathway
- sorting