The ubiquitin conjugation system is involved in ligand-induced endocytosis of the growth hormone receptor (GHR) via a cytosolic 10-amino acid ubiquitin-dependent endocytosis motif. Herein, we demonstrate that the proteasome is also involved in growth hormone receptor down-regulation. Ligand-induced degradation was blocked in the presence of specific proteasomal inhibitors. In addition, growth hormone (GH) internalization was inhibited, whereas the transferrin receptor cycle remained unaffected. A truncated GHR entered the cells independent of proteasome action. In addition, we show that GH internalization is independent of the presence of lysine residues in the cytosolic domain of the receptor, whereas its internalization can still be inhibited by proteasomal inhibitors. Thus, GHR internalization requires proteasome action in addition to an active ubiquitin conjugation system, but ubiquitination of the GHR itself seems not to be required. The growth hormone receptor (GHR)1 is a mammalian plasma membrane protein whose internalization is mediated by the ubiquitin conjugation system (1). In particular, a 10-amino acid motif including Phe-327 within the GHR cytosolic tail (the UbE motif) is involved in both GHR ubiquitination and ligand-induced receptor endocytosis. In addition, ubiquitination of the GHR itself is not necessary for ligand internalization (2). The receptor has a short half-life (3-5), and the degradation occurs within the lysosome (3, 6). However, it has been suggested that the GHR is also transported to the nucleus (7), to detergent-insoluble membrane domains (8), and back to the plasma membrane (9). GHR signaling is initiated at the plasma membrane when two receptors are dimerized by a single GH molecule (10). This dimerization induces recruitment and binding of the tyrosine kinase JAK2, resulting in the activation of various signal transduction pathways (reviewed in Ref. 11). The GHR was initially found to be ubiquitinated upon amino acid sequencing of the receptor from rabbit liver (12). Binding of GH stimulates ubiquitination, internalization, and degradation of the receptor. In a Chinese hamster cell line carrying a temperature-sensitive ubiquitin activation enzyme E1 (CHOts20; see Ref. 13), inactivation of E1 results in an accumulation of non-ubiquitinated GHRs at the plasma membrane, whereas internalization of the transferrin receptor is unaffected (1,14). These data show that GHR ubiquitination and internalization are related.Degradation of cytosolic proteins is mainly carried out by the 26 S proteasome. The ubiquitin conjugation system selects and targets the proteins for proteasomal degradation (15). In a growing number of cases the ubiquitin conjugation system seems to be involved in the selection steps directly preceding endocytosis at the plasma membrane. In yeast the ␣-factor receptor Ste2p (16), the Ste6 peptide transporter (17), Gap1p amino acid permease (18), Gal2p galactose transporter (19), Fur4 uracil permease (20), and Pdr5 (21), a multidrug transporter, all undergo ubiquitin-de...

The ubiquitin-proteasome pathway acts as a regulator of the endocytosis of selected membrane proteins. Recent evidence suggests that it may also function in the intracellular trafficking of membrane proteins. In this study, several models were used to address the role of the ubiquitin-proteasome pathway in sorting of internalized proteins to the lysosome. We found that lysosomal degradation of ligands, which remain bound to their receptors within the endocytic pathway, is blocked in the presence of specific proteasome inhibitors. In contrast, a ligand that dissociates from its receptor upon endosome acidification is degraded under the same conditions. Quantitative electron microscopy showed that neither the uptake nor the overall distribution of the endocytic marker bovine serum albumin-gold is substantially altered in the presence of a proteasome inhibitor. The data suggest that the ubiquitin-proteasome pathway is involved in an endosomal sorting step of selected membrane proteins to lysosomes, thereby providing a mechanism for regulated degradation.