J Biol Chem, Vol. 275, Issue 3, 1575-1580, January 21, 2000

Endocytosis and Degradation of the Growth Hormone Receptor Are Proteasome-dependent^*

Peter van Kerkhof, Roland Govers, Cristina M. Alves dos Santos, and Ger J. Strous^§

From the Department of Cell Biology, University Medical Center Utrecht and Institute of Biomembranes, Heidelberglaan 100, AZU-G02.525, 3584CX Utrecht, The Netherlands

	ABSTRACT

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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.

	INTRODUCTION

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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 (CHO-ts20; 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-dependent endocytosis. Inferred from genetic studies, proteasome activity is neither necessary for ubiquitin-dependent endocytosis nor for vacuolar degradation in yeast. In mammalian cells, studies with proteasomal inhibitors suggest a role for the proteasome in the degradation of the Met tyrosine kinase receptor (22), the platelet-derived growth factor receptor (23), the low density lipoprotein receptor (24), and the mannose phosphate receptor (25).

Down-regulation of signal transducing membrane receptors is a part of the highly programmed cascade of events leading both to extinction of the signaling pathway(s) and to rapid degradation of the primary messengers, the receptor and its ligand (5, 26-29). In the absence of ligand the half-life of GHR is approximately 1-2 h depending on the cell system used. The assumption is that this is mainly due to a proteolytic cleavage in the extracellular domain of the GHR resulting in soluble GH-binding proteins (30); the fate of the cytosolic tail in this process is unknown. If ligand is present, a completely different scenario follows; two GHR polypeptides dimerize, they are phosphorylated by the tyrosine kinase JAK2 and ubiquitinated, and the complex is then endocytosed. As the ubiquitin conjugation system acts generally in concert with the 26 S proteasome, we examined the effect of proteasomal inhibitors on GH uptake via wild-type GHR and a receptor truncated at amino acid residue 369 in CHO-ts20 cells (1). The data show that specific proteasomal inhibitors block GH uptake via the full-length GHR, whereas a truncated receptor can endocytose undisturbed. Nonetheless, the ubiquitin conjugation system remains necessary for the truncated receptor to be endocytosed (14). Evidence is provided that proteasomal action does not require ubiquitination of the receptor itself.

	MATERIALS AND METHODS

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Cells and Antibodies-- A polyclonal antibody to the cytosolic tail was raised in rabbits against the membrane-proximal amino acid residues 271-318 (anti-T) (see Fig. 1) as described in Ref. 1; antibody (Mab5) recognizing the luminal part of the GHR was from AGEN Inc., Parsippany, NJ. Antiserum specific for protein-ubiquitin conjugates was a generous gift from Dr. A. Ciechanover (Technion-Israel Institute of Technology, Haifa, Israel). CHO-ts20 was transfected with both the full-length rabbit GHR cDNA sequence and a cDNA encoding GHR truncation 1-369 (1, 13). 10 mM sodium butyrate was added to the cells 18 h before use to increase GHR expression (14).

Mutagenesis and Transfection-- cDNA encoding GHR truncation mutant GHR1-369 was constructed by introducing a stop codon at the proper position within the GHR cDNA. For this GHR truncation, a polymerase chain reaction was performed using a 5'-oligonucleotide containing a NcoI restriction site corresponding to the NcoI site in the cDNA of the transmembrane region of the GHR together with a 3'-oligonucleotide containing a KpnI restriction site, a stop codon, and overlapping sequences at position 369 within the cDNA, encoding the intracellular domain of the GHR. The polymerase chain reaction product was cut by NcoI and KpnI and ligated into a PGEM3Z-GHR construct. The truncated GHR cDNA (see Fig. 1) was subcloned into the pcDNA3 vector (Invitrogen). cDNA of mutants GHR F327A and GHR1-399 K271-362R were constructed as described (2, 14).

GH Binding and Internalization-- ¹²⁵I-human GH was prepared using chloramine T (1). For internalization studies, cells were grown in 35-mm dishes, washed with MEM supplemented with 20 mM Hepes and 0.1% bovine serum albumin, and incubated for 1 h at 30 °C in MEM/Hepes. ¹²⁵I-GH (8 nM) was bound on ice for 120 min in the absence or presence of excess unlabeled GH, and the cells were washed free of unbound GH and incubated for 0-30 min at 30 °C. If indicated, lactacystin (20 µM) or its -lactone (20 µM), MG132 (20 µM), and carboxybenzylleucyl-leucyl-leucinevinylsulfone (20 µM), dissolved in either ethanol or dimethyl sulfoxide, or vehicle only were added 1 h before the start of the experiment. Membrane-associated GH was removed by acid wash (0.15 M NaCl, 0.1% bovine serum albumin, 0.05 M glycine, pH 2.5), and internalized GH was determined by measuring the radioactivity after solubilization of the acid-treated cells by 1 M NaOH.

Metabolic Labeling-- For metabolic labeling, the cells were incubated in methionine-free MEM for 20 min and then [³⁵S]methionine (Tran-³⁵S Label^TM, 1.85 MBq/ml, 40 TBq/mmol, ICN Biomedicals, Costa Mesa, CA) was added and the incubation was continued for 20 min; the radioactivity was chased in the presence of MEM containing 0.05 mM unlabeled methionine, 0.18 µg/ml GH, 0.1% bovine serum albumin, and the appropriate proteasomal inhibitor. Cells were lysed in boiling buffer (see below). The radioactivity was determined using a Molecular Dynamics PhosphorImager.

Cell Lysis and Western Blotting-- At the end of the incubation, cells were washed and immediately lysed in boiling buffer containing 1% SDS in PBS. We used this protocol for all of our experiments to ascertain that no GHR or the derived degradation products were lost because of poor solubility of GH·GHR complexes, as reported by Goldsmith et al. (8). Equal aliquots of the cell extracts were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting as described (14). For detection we used the enhanced chemiluminescence system (Amersham Pharmacia Biotech).

Immunoprecipitations-- Immunoprecipitations were performed as described previously (1). Extracts from cells lysed in boiling buffer were subjected to immunoprecipitation in PBS containing 1% Triton X-100, 0.5% SDS, 0.25% sodium deoxycholate, 0.5% bovine serum albumin, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 2 µM MG132. The lysates were incubated with anti-ubiquitin or anti-GHR antiserum as indicated in the experiments represented in the figures for 2 h on ice. Immune complexes were isolated by the use of protein A-agarose beads (Repligen Co., Cambridge, MA).

Microscopy-- Cy3-GH and Cy3-transferrin were prepared using a FluoroLink Cy3 label kit according to the supplier's instructions (Amersham Pharmacia Biotech). Cells grown on coverslips were incubated for 60 min in MEM supplemented with 20 mM Hepes at 30 °C and for 30 min with Cy3-GH (1 µg/ml) or Cy3-transferrin (20 µg/ml). Cells were washed with PBS to remove unbound label and fixed for two h in 3% paraformaldehyde in PBS. Confocal laser scanning microscopy was performed using a Leica TCS 4D system.

	RESULTS

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Effect of Proteasomal Inhibitors on GHR Turnover-- Initially, we investigated the life cycle of the GHR at the permissive temperature (30 °C) in the presence of GH in CHO-ts20 cells transfected with GHR cDNA using pulse-chase labeling with [³⁵S]methionine (see Fig. 2A). The receptor is synthesized as a 110-kDa glycoprotein precursor (double band) and, upon "complex glycosylation" in the Golgi complex, converted to a 130-kDa mature species. The nature of the double band is unknown. Mature GHR was detectable by 20 min of chase and was maximal at 80 min, after which rapid degradation occurred. When the pulse-chase was performed in the presence of the specific proteasomal inhibitors MG132 or the more membrane permeable -lactone analogue of lactacystin (31, 32), the amount of labeled mature GHR was hardly decreased after 160 min of chase. Both inhibitors did not affect the endoplasmic reticulum to Golgi transport, as is concluded from the kinetics by which the GHR precursor is converted to the mature GHR. When the experiment was performed with a cell line expressing the GHR with the cytosolic tail truncated after amino acid residue 369 (GHR1-369, Fig. 1), basically the same results were obtained (Fig. 2B). In the presence of clasto-lactacystin -lactone the amount of GHR1-369 that became mature after 80 min was diminished, but the kinetics of degradation between 80 and 160 min of chase were similar to the results with MG132. These results demonstrate that the mature GHR has a relatively short half-life and that the proteasome plays an important role in GH-induced degradation.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the wild-type and mutant GHRs. For GHR mutants truncated at residue 399, all cytoplasmic lysines, lysine mutations, and Phe-327 are indicated. For GHR mutants truncated at residue 369, only Phe-327 is indicated. A black square represents the transmembrane domain. Hatched squares represent box-1 (site for JAK2 binding) and the UbE motif (site for interaction with the ubiquitin system). The antibody specificities are indicated at the top of the figure. Mab5 is a monoclonal antibody raised against GH-binding proteins; anti-T was raised against a GST-fusion protein to the GHR peptide 271-318.

View larger version (108K): [in this window] [in a new window]   Fig. 2.   Effect of proteasomal inhibitors on [35S]methionine-labeled GHRs. CHO-ts20 cells were labeled with [35S]methionine for 20 min and chased in MEM supplemented with 0.1% bovine serum albumin and 0.18 µg/ml GH for the time periods indicated. GHR was immunoprecipitated using anti-T. MG132 or -lactone were present 60 min before the start and throughout the pulse-chase period. A, CHO-ts20 cells expressing the full-length GHR. m, mature (130 kDa of GHR); p, precursor (110 kDa). B, CHO-ts20 cells expressing the GHR1-369. m, mature (85 kDa of GHR); p, precursor (60 kDa). Relative molecular weight standards (Mr × 103) are shown to the right. The amounts of radioactivity were determined using ImageQuant (Molecular Dynamics) and were expressed as percentages of the radioactivity incorporated in the precusor GHR after the pulse labeling. , precursor GHR; , mature GHR. The values represent the mean ± S.D. of two different experiments.

Effect of Proteasomal Inhibitors on GHR Endocytosis-- Ligand-induced endocytosis of the growth hormone receptor occurs only if the ubiquitin conjugation system is functional. An issue is thus whether at the same time proteasomal action is required. To test this we used the GHR-transfected CHO-ts20 cells expressing approximately 10⁶ ligand-binding sites per cell (14). Incubation for 30 min in the presence of Cy3-labeled GH resulted in abundant fluorescent label in endosomal and lysosomal compartments (Fig. 3A). If the cells were treated with MG132 (Fig. 3D) or lactacystin (Fig. 3G) little label was present intracellularly. The same results were obtained when the cells were treated with carboxybenzyl-leucyl-leucyl-leucyl vinylsulfone or clasto-lactacystin -lactone (not shown). To ascertain that these proteasomal inhibitors did not cause pleiotropic effects on the receptor-mediated endocytic machinery we used Cy3-labeled transferrin under identical conditions (Fig. 3, C, F, and I); no inhibition of transferrin uptake was observed. We next addressed the question as to whether the proteasome acts directly or indirectly on the GHR. Experiments with Chinese hamster cells expressing a GHR1-369 show that the same proteasomal inhibitors as used for the full-length GHR do not affect GH endocytosis (Fig. 3, B, E, and H). Thus, removal of a portion of the cytosolic tail is sufficient to uncouple proteasomal action and GH-dependent endocytosis.

View larger version (159K): [in this window] [in a new window]   Fig. 3.   Effect of proteasomal inhibitors on Cy3-GH and Cy3-transferrin endocytosis. CHO-ts20 cells, expressing either wild-type (A, D, G, C, F, and I), or truncated GHR1-369 (B, E, and H) were incubated with vehicle (A-C), 20 µM MG132 (D-F), or 20 µM lactacystin (G-I) for 1 h at 30 °C; then Cy3-GH (A, B, D, E, G, and H) or Cy3-transferrin (C, F, and I) were added for 30 min, and the cells were washed, fixed, and the fluorescence was visualized by confocal microscopy. No uptake was observed when excess unlabeled ligand was added. Virtually no label was visible when the cells in D and G were treated at pH 2.5 before fixation (not shown).

To confirm and quantify the effect of the proteasomal inhibitors we measured the uptake of ¹²⁵I-GH in kinetic experiments (Fig. 4). Cells were pre-treated with the inhibitors, ¹²⁵I-GH was bound on ice, and cells were incubated at 30 °C for various periods of time. After cooling to 0 °C and washing the cells at a low pH value to remove label from the cell surface, uptake was determined. Again, GH uptake was inhibited by MG132. To avoid secondary effects such as receptor recycling and/or early ligand degradation, we measured the initial uptake kinetics. To ascertain the involvement of proteasomes in this process we used several other proteasomal inhibitors; lactacystin reduced the uptake somewhat less effectively, probably because of its poor cell-permeant properties, whereas clasto-lactacystin -lactone was almost as effective as MG132 (Fig. 4, lower panel). As expected, uptake via the GHR1-369 was not affected by the proteasomal inhibitors. It was also observed that the truncated receptor is internalized much more rapidly than the full-length GHR. To determine background uptake values we used the full-length GHR in which the Phe-327 was mutated to alanine. This mutation abolishes the ubiquitin-dependent uptake of GH. As seen in Fig. 4 (GHR-F327A), this receptor shows a low GH uptake that was not further decreased in the presence of MG132. The level of GH uptake via the GHR F327A mutant is similar to that via the wild-type GHR in the presence of MG132, indicating that proteasomal inhibitors affect the ubiquitin system-dependent GH uptake. These results show that ubiquitin system-dependent uptake via the full-length receptor is fully blocked in the presence of proteasomal inhibitors.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effect of proteasomal inhibitors on uptake kinetics of 125I-GH. CHO-ts20 cells were incubated with or without inhibitors for 1 h at 30 °C and put on ice for 2 h with 125I-GH. The cells were then incubated at 30 °C as indicated. Background label was determined in the presence of excess unlabeled GH and subtracted. The amounts of internalized 125I-GH are plotted as a percentage of the cell-associated radioactivity at the start of incubation. For the experiments in the lower panel 125I-GH uptake was measured without prior binding on ice; this explains the different uptake kinetics as compared with the upper panels. , control (1% ethanol); , MG132; , lactacystin, X, clasto-lactacystin -lactone.

Ubiquitination of the GHR Itself Is Not Required-- To investigate the role of ubiquitination in the proteasome-dependent endocytosis of the GHR, we addressed whether ubiquitination of the receptor was required. We constructed a GHR in which all cytoplasmic lysine residues were mutated to arginines. Because internalization of ligand by GHR1-399 is as dependent on an intact ubiquitin conjugation system as the wild-type GHR, we used a construct for these experiments in which all 10 cytoplasmic lysine residues were mutated (GHR1-399, K271-362R) (18). Both truncations endocytose GH in an ubiquitin system-dependent fashion (2). Fig. 5 shows that MG132 does inhibit ligand uptake for the GHR1-399. In addition it shows that, even if the GHR tail does not contain attachment sites for ubiquitin, endocytosis is inhibited by the proteasomal inhibitor. To quantify the fluorescence experiments ¹²⁵I-GH uptake was measured in these cells in the presence and absence of the proteasomal inhibitor MG132. Although the inhibitory effect of MG132 is less than for the full-length GHR, it is clear that MG132 inhibits ligand uptake (Fig. 6). An important difference between GH-uptake by the full-length and the truncated GHR is that the initial rate of uptake is higher as the cytosolic tail is shorter. This is certainly the case for truncations up to the di-leucine motif at position 349 (33). An explanation is still nonexistent. To show that ubiquitin attachment is indeed absent, we immunoprecipitated the two truncated GHRs with an antiserum against ubiquitin after stimulation with GH for 30 min and detected the blots with anti GHR. As seen in Fig. 7, GH induces a strong increase in the amount of ubiquitinated GHR1-399, whereas no signal is detectable for the lysine-less GHR1-399. The amounts of mature receptors in both cell lines is approximately the same, as shown in Fig. 7 (right panel), in which cell lysates were immunoblotted with anti-GHR (anti-T). These results allow two conclusions: 1) the inhibitory effect of proteasomal inhibitors resides in a 30-amino acid segment between amino acid residues 369 and 399, and 2) ubiquitination of the (truncated) GHR is not required for the proteasome-dependent GHR internalization.

View larger version (122K): [in this window] [in a new window]   Fig. 5.   Internalization of ligand by a GHR that contains no potential ubiquitin conjugation sites. CHO-ts20 cells expressing GHR truncation mutant 1-399 or truncation 1-399 K271-362R were incubated with Cy3-GH for 1 h at 30 °C without (Control) or with MG132. Cy3-GH was visualized by confocal microscopy.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Quantitation of GH-uptake by lysine-less GHR. For details, see the legend to Fig. 4.

View larger version (76K): [in this window] [in a new window]   Fig. 7.   Ubiquitination assay of the lysine-less GHR. Cells expressing GHR1-399 or the lysine-less counterpart (GHR1-399, K271-362R, designated as GHR1-399K) were incubated with or without GH for 30 min at 30 °C. Left panel, immunoprecipitations (IP) were performed with anti-ubiquitin and analyzed by immunoblotting (Blot) using anti-GHR (Mab5). Bracket indicates ubiquitinated GHR; p, precursor GHR1-399; m, mature GHR1-399. Right panel, cell lysates (equal amounts of protein) were immunoblotted with anti-T.

	DISCUSSION

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It is generally accepted that the ubiquitin/proteasome system is involved in selective degradation of cytosolic and nuclear proteins (15). At the cytosolic face of the endoplasmic reticulum the ubiquitin/proteasome system is involved in degradation of misfolded endoplasmic reticulum proteins (34, 35). Previously, we have shown that GHR endocytosis requires an intact ubiquitin system and that GH internalization is accompanied by GHR ubiquitination (1, 14). Endocytosis of the GHR occurs via clathrin-coated pits (28). This process requires interaction between adapter proteins such as AP2 or -arrestin and clathrin as well as endocytosis motifs within the cytosolic tail of membrane proteins. The tyrosine-based motif YXX (where X is any amino acid and  is an amino acid with a bulky hydrophobic group) is involved in the endocytosis of many transmembrane proteins such as the receptors for low density lipoprotein, transferrin, and asialoglycoproteins (36-38). Internalization of the insulin and 2-adrenergic receptors is mediated by the di-leucine endocytosis motif (39, 40). GHR contains an endocytosis-competent di-leucine motif (DTDRLL), but this acts only if the receptor is truncated immediately after the motif (33). Moreover, ligand-induced GHR endocytosis fully depends on the UbE motif (2). Herein we present evidence that proteasomal action is required for receptor down-regulation in addition to the ubiquitin system and the clathrin-coated pit machinery.

What is the substrate for the proteasome? The most obvious target is the GHR tail itself. This is indicated by the fact that a GHR truncated at position 369 can enter the cells if proteasomal action is blocked. In this scenario the GHR can only be recognized by the endocytic machinery if the tail is cut. In favor of this idea is the fact that attempts to detect full-length GHR intracellularly failed (not shown). Another possibility is that the GHR binds, via the amino acid sequence 369-399, to a factor that has to be removed by the proteasome before endocytosis can proceed. This would explain why ubiquitination of the GHR per se is not required.

The amino acid sequence starting at amino acid 365 (DSGRTS) is homologous to an amino acid sequence of human immunodeficiency virus, type I-encoded gene, the membrane protein Vpu. Vpu is a target for the ubiquitin system in the rough endoplasmic reticulum and acts as an intermediary in the degradation of CD4 (41). In the truncated receptor GHR1-369 this motif is disrupted, which would provide a third possible scenario: the F-box protein TrCP or an analogous protein would bind via its WD40 domain to the motif in the full-length receptor, after which proteasomal action could start. Whatever scenario is correct, it is unlikely that the proteasome truncates the receptor beyond residue 334, because this would disable the UbE motif and prevent endocytosis (14).

Ubiquitination of the GHR is not required for proteasomal action. This phenomenon was reported before for GHR endocytosis via the UbE motif. If truncated receptors like GHR1-369 and GHR1-334 are used endocytosis still depends on an active ubiquitin conjugation system, but their ubiquitination is not required (2). Moreover, proteasomal inhibitors do not affect GH uptake via these short GHRs (Fig. 4 and data not shown). Thus, both proteasomal action and UbE-induced events precede endocytosis (i.e. selection into the coated pits and coated vesicle formation). Collectively, the data indicate that the action of proteasomes precedes the events directed via the UbE motif, although it cannot be excluded that the latter might control the action of the proteasome. For both events, ubiquitination of the GHR tail is not required. A possible explanation is that the ubiquitin ligases involved contain ubiquitin-like proteins as reported for the E3 that is involved in von Hippel-Lindau tumor suppressor function (42).

Once established that proteasomal inhibitors prevent endocytosis, it is not surprising that the half-life of the GHR is prolonged under such conditions. Previously we and others have shown that the GHR and its ligand are degraded within the lysosome (1, 3, 6). This is certainly the case for the luminal domain of the receptor. Our present data strongly suggest that at least a portion of the GHR is a target for the proteasome system. It has been proposed that the lysosomal and the ubiquitin/proteasome pathway may cooperate in degrading some tyrosine kinase receptors (23). Proteasomal action has been reported to be involved in the turnover of other receptors like the Met tyrosine kinase receptor (22), the platelet-derived growth factor- receptor (43), the low density lipoprotein receptor (24), and the mannose phosphate receptor (25). Endocytosis of the latter three receptors is not dependent on the ubiquitin/proteasome system. More similarities exist between the degradation of the Met tyrosine kinase receptor and the GHR. Like the GHR, the Met tyrosine kinase receptor undergoes a ligand-independent proteolytic cleavage in its extracellular domain (44), and the receptor is ubiquitinated upon ligand binding (22). Our observation that specific proteasomal inhibitors inhibit degradation of wild-type GHR (at the cell surface) and truncated GHR1-369 (at the cell surface and in endosomes) suggests that the proteasome inhibitors affect both endocytosis and membrane sorting to the lysosomes. One striking difference between the two receptors is that ubiquitination of the Met receptor is increased in the presence of proteasomal inhibitors, whereas ubiquitination of the GHR is decreased under all conditions that block its endocytosis (14), including proteasomal inhibitors (not shown). As the Met tyrosine kinase receptor lacks the UbE motif that is instrumental in ubiquitin-dependent GHR endocytosis, it is tempting to speculate that stabilization of the Met tyrosine kinase receptor by proteasomal inhibitors is due to an intracellular block e.g. in a membrane-sorting step en route to the lysosomes, analogous to the mechanism that connects cbl to the degradation of epidermal growth factor and platelet-derived growth factor receptors (45, 46).

Although the present findings apply to the GHR, there are indications that the ubiquitin/proteasome system is involved in regulation of the residence time at the cell surface of other membrane proteins as well. The residence time of the sodium channel protein ENaC is regulated by the ubiquitin system (47). Many signaling membrane receptors e.g. the Met tyrosine kinase receptor (22), the TCR -chain (48), the c-Kit receptor (49), epidermal growth factor receptor (50), and prolactin receptor (51) are all ubiquitinated upon activation. Thus, the ubiquitin/proteasome system may set the timer for cell surface residency and life time for a selected number of key regulatory cell surface molecules.

	ACKNOWLEDGEMENTS

We thank Drs. Alan Schwartz and Guojun Bu for stimulating discussions, Dr. Hidde Ploegh for the proteasomal inhibitors, Dr. William Wood (Genentech) for kindly providing the GHR cDNA, and Eli Lilly for the kind gift of GH.

	FOOTNOTES

^* This work was supported by Grants NWO-902-23-188 and NWO-902-68-244 from the Netherlands Organization for Scientific Research and by European Union Network Grant ERBFMRXCT96-0026.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Nephrology and Hypertension, AZU, Utrecht, The Netherlands.

^§ To whom correspondence should be addressed. Tel.: 31-30-2506476; Fax: 31-30-2541797; E-mail: strous@med.uu.nl.

	ABBREVIATIONS

The abbreviations used are: GHR, growth hormone receptor; GH, growth hormone; CHO-ts20, Chinese hamster cell line carrying a temperature-sensitive ubiquitin activation enzyme E1; MEM, minimum Eagle's medium; PBS, phosphate-buffered saline.

	REFERENCES

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