Ubiquitination-independent Trafficking of G Protein-coupled Receptors to Lysosomes

Abstract

Ubiquitination of cytoplasmic lysine residues can target G protein-coupled receptors (GPCRs) to proteasomes and has recently been shown to also be required for sorting of certain GPCRs to lysosomes following ligand-induced endocytosis. We addressed the generality of this mechanism by examining regulated proteolysis of the murine ␦ opioid receptor (DOR) expressed in human embryonic kidney 293 cells, a well characterized model system in which receptors are sorted to lysosomes. Incubation of cells in the presence of the highly specific proteasome inhibitor lactacystin did not detectably affect ligand-induced proteolysis of DOR but significantly delayed ligand-induced proteolysis of epidermal growth factor receptors. Mutation of all cytoplasmic lysine residues in DOR, creating a mutant opioid receptor that is unable to be ubiquitinated, did not detectably inhibit either ligand-induced endocytosis or proteolytic degradation of endocytosed receptors. Furthermore, the lysinemutated DOR, like its wild type counterpart, colocalized extensively with lysosomes after ligand-induced endocytosis. These results demonstrate that ubiquitination of DOR is not required either for its ligand-induced endocytosis or for postendocytic trafficking to lysosomes.Classical studies of receptor-mediated endocytosis established the principle of geometric sorting whereby membranebound receptors recycle to the plasma membrane by bulk membrane flow and require specific membrane sorting machinery to traffic to lysosomes (1-3). Although many signaling receptors are endocytosed after ligand-induced activation, there are large differences in the degree to which various receptors thereafter traffic to lysosomes. Such differences in the endocytic trafficking of receptors have important functional consequences (4 -6). In the case of G protein-coupled receptors (GPCRs), 1 events surrounding the initial endocytosis of receptors generally result in a rapid desensitization of signaling activity. Recycling of receptors back to the plasma membrane typically promotes functional resensitization, whereas sorting of internalized receptors to lysosomes leads to proteolytic down-regulation of receptors and a prolonged attenuation of signal transduction (7-9). These considerations have motivated considerable interest in elucidating mechanisms that control the sorting of specific signaling receptors in the endocytic pathway. Covalent modification of lysine residues by ubiquitin is well known to function as a signal targeting many proteins to proteasomes. Ubiquitination of cytoplasmic lysine residues has emerged more recently as a mechanism that also regulates the endocytic trafficking of various integral membrane proteins (10). Ubiquitination of lysine residues located in the carboxylterminal cytoplasmic domain of the yeast GPCR Ste2p was initially shown to promote ligand-induced endocytosis of receptors (11). More recently, ubiquitination of the cytoplasmic tail of Ste2p (12) has been shown to be essential for the subsequent sorting of endocytosed receptors to int...

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Resistance of the Human β1-Adrenergic Receptor to Agonist-induced Ubiquitination

“…Proteasomal inhibitors are reported to either reduce (12) or have no effect (17) on agonist-mediated internalization and degradation of ␤ 2 AR. In one study proteasomal but not lysosomal inhibitors reduced the down-regulation of and ␦ opioid receptors (14), whereas in another, lactacystin, a very specific proteasomal inhibitor, did not impair agonist-induced internalization, lysosomal targeting, and degradation of the ␦ opioid receptor (15). Some of these studies found that steadystate receptor levels increase in unstimulated cells treated with proteasomal inhibitors (14,17).…”

“…14, but see Ref. 15), and the vasopressin V2 receptor (16). Eliminating receptor ubiquitination reduces agonist-mediated receptor degradation but not internalization.…”

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