The ubiquitin‐dependent protein degradation system has recently been implicated in downregulation of signal transducing receptors. Growth hormone receptor (GHR) cDNA was transfected into Chinese hamster ovary cells, which exhibit a temperature‐sensitive defect in ubiquitin conjugation (CHO‐ts20), as well as into wild‐type cells (CHO‐E36). Upon binding of growth hormone (GH), two GHR polypeptides dimerize and initiate signal transduction. In CHO‐E36 and in CHO‐ts20 at the permissive temperature the GHR was ubiquitinated and degraded in a GH‐dependent fashion. However, at the non‐permissive temperature in CHO‐ts20 cells, neither GH‐dependent uptake nor degradation of the GHR was observed, while in CHO‐E36 cells both GHR uptake and degradation were accelerated. Incubation of CHO‐E36 cells with inhibitors of endosomal/lysosomal function (NH4Cl, bafilomycin A1) markedly reduced ligand‐induced GHR degradation. Our results indicate that a functional ubiquitin conjugating system is required for GH‐induced endocytosis and that degradation of both the exoplasmic and cytoplasmic portions of the GHR occurs within the endosomal/lysosomal compartment.

All members of the low density lipoprotein (LDL) receptor family contain at least one copy of the NPXY sequence within their cytoplasmic tails. For the LDL receptor, it has been demonstrated that the NPXY motif serves as a signal for rapid endocytosis through coated pits. Thus, it is generally believed that the NPXY sequences function as endocytosis signals for all the LDL receptor family members. The primary aim of this study is to define the endocytosis signal(s) within the cytoplasmic tail of LDL receptor-related protein (LRP). By using LRP minireceptors, which mimic the function and trafficking of full-length endogenous LRP, we demonstrate that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We also found that the distal di-leucine motif within the LRP tail contributes to its endocytosis, and its function is independent of the YXXL motif. Although the proximal NPXY motif and the proximal di-leucine motif each play a limited role in LRP endocytosis in the context of the full-length tail, these motifs were functional within the truncated receptor tail. In addition, we show that LRP minireceptor mutants defective in endocytosis signal(s) accumulate at the cell surface and are less efficient in delivery of ligand for degradation.

The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multiligand endocytic receptor and a member of the LDL receptor family. Here we show that sorting nexin 17 (Snx17) is part of the cellular sorting machinery that regulates cell surface levels of LRP by promoting its recycling. While the phox (PX) domain of Snx17 interacts with phosphatidylinositol-3-phosphate for membrane association, the FERM domain and the carboxyl-terminal region participate in LRP binding. Immunoelectron microscopy shows that the membrane-bound fraction of Snx17 is localized to the limiting membrane and recycling tubules of early endosomes. The NPxY motif, proximal to the plasma membrane in the LRP cytoplasmic tail, is identified as the Snx17-binding motif. Functional mutation of this motif did not interfere with LRP endocytosis, but decreased LRP recycling from endosomes, resulting in increased lysosomal degradation. Similar effects are found after knockdown of endogenous Snx17 expression by short interfering RNA. We conclude that Snx17 binds to a motif in the LRP tail distinct from the endocytosis signals and promotes LRP sorting to the recycling pathway in the early endosomes.

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...