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

Abstract

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.

Figure 1. Major GPCR signaling responses and regulation by endocytosis

A. Simplified schematic depicting the range of signaling responses mediated by GPCRs in mammals. B. Major trafficking itineraries of GPCRs that have been linked to functional regulation of cellular signaling. Endocytosis of receptors can produce functional desensitization of cellular responsiveness by physically removing receptors from access to extracellular ligands, and preventing access to plasma membrane-delimited signaling mediators. Recycling of internalized receptors can function, conversely, to resensitize cellular responsiveness. Trafficking of internalized receptors via the MVB/lysosome pathway causes a prolonged attenuation of cellular responsiveness by promoting proteolytic downregulation of receptors.

Figure 2. Comparison of regulated endocytosis of GPCRs in budding yeast and mammalian cells

A. Ligand-induced endocytosis of Ste2p in budding yeast is initiated by receptor phosphorylation by Yck2p, a casein kinase I -like enzyme, which promotes lysyl-ubiquitination of the cytoplasmic tail by Rsp5p. Ubiquitination is thought to promote endocytosis by linking receptors to the epsins Ent1p and 2p, and interaction with the Eps15 homologue Ede1p, all of which contain ubiquitin-binding domains. As discussed in the text, ubiquitination of Ste3p is required for constitutive receptor endocytosis but not for the ligand-induced component. B. Regulated endocytosis of many mammalian GPCRs is initiated by ligand-induced phosphorylation of receptors by a family of GPCR kinases (GRKs), which promote receptor interaction with the endocytic adaptor proteins β-arrestin-1 and -2 (arrestin 2 and 3). This mechanism does not require ubiquitination of the GPCR although, as discussed in the text, it can be influenced by ubiquitination of arrestin.

Figure 3. Mechanisms implicated in sorting mammalian GPCRs between recycling and lysosomal itineraries

Mechanism (1) depicts the canonical ubiquitin and ESCRT-dependent mechanism, similar to that described in yeast and for the EGF receptor tyrosine kinase in mammalian cells. This mechanism is proposed to mediate ubiquitination-dependent lysosomal sorting of a number of mammalian GPCRs, as discussed in the text. Mechanism (2) depicts proposed roles of additional machinery effectively upstream of the ESCRT, as demonstrated for several mammalian GPCRs including the delta opioid receptor. Receptor interactions with GASP, and possibly also with arrestins, inhibits receptor recycling and promotes traffic to the late endocytic pathway. Efficient recycling of receptors requires an active sorting process mediated by receptor interaction with distinct cellular proteins such as sorting nexin 27 (SNX27). None of these sorting steps requires receptor ubiquitination but, for receptors directed to the late endocytic pathway by GASPs, later steps in the proteolytic pathway are ESCRT-dependent and regulated by receptor ubiquitination. Mechanism (3) illustrates an alternate mechanism of GPCR trafficking to lysosomes, proposed for PAR1, which does not require receptor ubiquitination or the ESCRT and instead requires sorting nexin 1 (SNX1).