The novel ALIX-dependent GPCR sorting pathway is regulated by the a-arrestin ARRDC3. A critical role is also shown for the E3 ubiquitin ligase WWP2 in regulation of ALIX ubiquitination and lysosomal sorting of GPCRs.

Endocytic sorting and lysosomal degradation are integral to the regulation of G protein-coupled receptor (GPCR) function. Upon ligand binding, classical GPCRs are activated, internalized and recycled or sorted to lysosomes for degradation, a process that requires receptor ubiquitination. However, recent studies have demonstrated that numerous GPCRs are sorted to lysosomes independent of receptor ubiquitination. Here, we describe an ubiquitin-independent lysosomal sorting pathway for the purinergic GPCR P2Y1. After activation, P2Y1 sorts to lysosomes for degradation independent of direct ubiquitination that is mediated by a YPX3L motif within the second intracellular loop that serves as a binding site for the adaptor protein ALIX. Depletion of ALIX or site-directed mutation of the YPX3L motif inhibits P2Y1 sorting into the lumen of multivesicular endosomes/lysosomes and degradation. These findings confirm the function of YPX3L motifs as lysosomal targeting sequences for GPCRs and demonstrate that ALIX mediates the ubiquitin-independent degradation of certain GPCRs.

1. We studied the immediate and short-term effects of UV light in the near-visible range at the cellular and membrane level using the whole-cell patch-clamp technique in combination with digital fluorescence imaging. 2. Illumination with monochromatic UVA light (340-380 nm) induced a sustained non-saturable increase in membrane conductance dependent on wavelength and light intensity in several different mammalian cell types including RBL, mast, HEK, PC12 and 3T3 cells. 3. The current was non-selective for cations and permeable to Ca2+, but was inhibited by trivalent cations and was not due to the activation of an endogenous ion channel. We termed this novel current ILiNC for light-induced non-selective cation current. 4. A similar current was evoked by chemical peroxidants such as hydrogen peroxide and tertbutylhydroperoxide, but not by cytosolic oxidized glutathione. 5. The free-radical scavengers tocopherol (vitamin E) and ascorbic acid (vitamin C) significantly reduced the UV light effect. 6. The generation of the current was membrane delimited since it could be induced by the same UVA treatment in cell-free membrane patches showing a similar wavelength dependence. 7. These results suggest that ILiNC is activated by UVA light-induced generation of free radicals acting through lipid or protein peroxidation, and may represent a ubiquitous mechanism by which Na+ and Ca2+ can enter cells after phototoxic or free radical-induced membrane damage.

No abstract

Photomodification of the plasma membrane by visible light in the presence of the membrane-active sensitizer photofrin II is of considerably greater consequence for the electrical properties of the membrane, compared with exposure to ionizing radiation of the membrane and its aqueous environment. The differences are thought to reflect the types of reactive species produced by the two methods and their site of generation.