James N. Hislop

Role of Mammalian Vacuolar Protein-sorting Proteins in Endocytic Trafficking of a Non-ubiquitinated G Protein-coupled Receptor to Lysosomes

Many signaling receptors require covalent modification by ubiquitin for agonist-induced down-regulation via endocytic trafficking to lysosomes, a process that is mediated by a conserved set of endosome-associating proteins also required for vacuolar protein-sorting (VPS) in yeast. The delta opioid receptor (DOR) is a G protein-coupled receptor that can undergo agonist-induced proteolysis via endocytic trafficking to lysosomes but does not require covalent modification by ubiquitin to do so. This raises the question of whether lysosomal down-regulation of this “ubiquitination-independent” GPCR is mediated by a completely distinct biochemical mechanism or if similar VPS machinery is involved. Agonist-induced proteolysis of DOR was significantly inhibited by dominant negative mutant versions of Vps4/Skd1, an AAA-family ATPase required for a late step in lysosomal sorting of ubiquitinated membrane cargo. Furthermore, overexpression and interfering RNA-mediated knockdown indicated that lysosomal trafficking of opioid receptors is also dependent on Hrs, a VPS protein that mediates an early step in lysosomal sorting of ubiquitinated cargo. However, interfering RNA-mediated knockdown of Tsg101, a VPS protein that is essential for an intermediate step of the conserved lysosomal sorting mechanism, did not detectably affect agonist-induced proteolysis of DOR in the same cells in which (ubiquitination-dependent) lysosomal trafficking of epidermal growth factor receptors was clearly inhibited. These results indicate that opioid receptors, despite their ability to undergo efficient agonist-induced trafficking to lysosomes in the absence of covalent modification by ubiquitin, utilize some (Vps4 and Hrs) but perhaps not all (Tsg101) of the VPS machinery required for lysosomal sorting of ubiquitinated membrane cargo.

Role of ubiquitination in endocytic trafficking of G-protein-coupled receptors.

Lysyl ubiquitination has long been known to target cytoplasmic proteins for proteasomal degradation, and there is now extensive evidence that ubiquitination functions in vacuolar/lysosomal targeting of membrane proteins from both the biosynthetic and endocytic pathways. G-protein-coupled receptors (GPCRs) represent the largest and most diverse family of membrane proteins, whose function is of fundamental importance both physiologically and therapeutically. In this review, we discuss the role of ubiquitination in the vacuolar/lysosomal downregulation of GPCRs through the endocytic pathway, with a primary focus on lysosomal trafficking in mammalian cells. We will summarize evidence indicating that mammalian GPCRs are regulated by ubiquitin-dependent mechanisms conserved in budding yeast, and then consider evidence for additional ubiquitin-dependent and -independent regulation that may be specific to animal cells.

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 δ-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 δ-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 receptors hydrophobic core.

Regulation of Endocytic Clathrin Dynamics by Cargo Ubiquitination

Summary Some endocytic cargoes control clathrin-coated pit (CCP) maturation, but it is not known how such regulation is communicated. We found that μ-opioid neuropeptide receptors signal to their enclosing CCPs by ubiquitination. Nonubiquitinated receptors delay CCPs at an intermediate stage of maturation, after clathrin lattice assembly is complete but before membrane scission. Receptor ubiquitination relieves this inhibition, effectively triggering CCP scission and producing a receptor-containing endocytic vesicle. The ubiquitin modification that conveys this endocytosis-promoting signal is added to the receptor’s first cytoplasmic loop, catalyzed by the Smurf2 ubiquitin ligase, and coordinated with activation-dependent receptor phosphorylation and clustering through Smurf2 recruitment by the endocytic adaptor beta-arrestin. Epsin1 detects the signal at the CCP and is required for ubiquitin-promoted scission. This cargo-to-coat communication system mediates a biochemical checkpoint that ensures appropriate receptor ubiquitination for later trafficking, and it controls specific receptor loading into CCPs by sensing when a sufficient quorum is reached. Video Abstract

The role of ubiquitination in lysosomal trafficking of δ-opioid receptors

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.

Alternative splicing determines the post-endocytic sorting fate of G protein-coupled receptors

Mu-type opioid receptors are physiologically important G-protein-coupled receptors that are generally thought to recycle after agonist-induced endocytosis. Here we show that several alternatively spliced receptor variants fail to do so efficiently because of splice-mediated removal of an endocytic sorting sequence that is present specifically in the MOR1 variant. All of the recycling-impaired receptor variants were found to undergo proteolytic down-regulation more rapidly than MOR1, irrespective of moderate differences in endocytic rate, indicating that alternative splicing plays a specific role in distinguishing the trafficking itinerary of receptors after endocytosis. The recycling-impaired MOR1B variant was similar to MOR1 in its ability to mediate opioid-dependent inhibition of adenylyl cyclase, and to undergo opioid-induced desensitization in intact cells. Functional recovery (resensitization) of MOR1B-mediated cellular responsiveness after opioid removal, however, was significantly impaired (4-fold reduction in rate) compared with MOR1. To our knowledge the present results are the first to establish a role of alternative RNA processing in specifying the post-endocytic sorting of G-protein-coupled receptors between divergent and functionally distinct membrane pathways.

Ubiquitination in the First Cytoplasmic Loop of μ-Opioid Receptors Reveals a Hierarchical Mechanism of Lysosomal Down-regulation

Background: It is not known if ubiquitination controls endocytic trafficking of μ-opioid receptors. Results: Endocytic sorting to the proteolytic compartment is ubiquitination-independent. Ubiquitination specifically in the first cytoplasmic loop promotes multivesicular body sorting required for subsequent destruction of the ligand binding site. Conclusion: The ubiquitinated first cytoplasmic loop represents a discrete downstream sorting determinant. Implications: The down-regulation mechanism is hierarchical. μ-Type opioid receptors (MORs) are members of the large seven-transmembrane receptor family which transduce the effects of both endogenous neuropeptides and clinically important opioid drugs. Prolonged activation of MORs promotes their proteolytic degradation by endocytic trafficking to lysosomes. This down-regulation process is known to contribute to homeostatic regulation of cellular opioid responsiveness, but mechanisms that mediate and control MOR down-regulation have not been defined. We show here that lysosomal down-regulation of MORs is ESCRT (endosomal sorting complex required for transport)-dependent and involves ubiquitin-promoted transfer of internalized MORs from the limiting endosome membrane to lumen. We also show that MOR down-regulation measured by conventional radioligand binding assay is determined specifically by ubiquitination in the first cytoplasmic loop. Surprisingly, we were unable to find any role of ubiquitination in determining whether internalized receptors recycle or are delivered to lysosomes. Instead, this decision is dictated specifically by the MOR C-tail and occurs irrespectively of the presence or absence of receptor ubiquitination. Our results support a hierarchical organization of discrete ubiquitin-independent and -dependent sorting operations, which function non-redundantly in the conserved down-regulation pathway to mediate precise endocytic control. Furthermore, they show that this hierarchical mechanism discriminates the endocytic regulation of naturally occurring MOR isoforms. Moreover, they are the first to reveal, we believe, for any seven-transmembrane receptor, a functional role of ubiquitination in the first cytoplasmic loop.

Analysis of GPCR localization and trafficking.

Localization and trafficking of G protein-coupled receptors (GPCRs) is increasingly recognized to play a fundamental role in receptor-mediated signaling and its regulation. Individual receptors, including closely homologous subtypes with otherwise similar functional properties, can differ considerably in their membrane trafficking properties. In this chapter, we describe several approaches for experimentally assessing the subcellular localization and trafficking of selected GPCRs. Firstly, we describe a flexible method for receptor localization using fluorescence microscopy. We then describe two complementary approaches, using fluorescence flow cytometry and surface biotinylation, for examining receptor internalization and trafficking in the endocytic pathway.

Regulated Membrane Trafficking and Proteolysis of GPCRs

Multiple mechanisms contribute to the physiological regulation of G protein-coupled receptors (GPCRs) present in the plasma membrane, the main site where ligand-induced signaling events are initiated. Early studies delineated the existence of distinct functional processes of receptor regulation in natively expressing cells and tissues (1,2). More recent studies have led to an explosion of new information regarding cellular and molecular mechanisms of receptor regulation.