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.