| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
GRH-SOMATOSTATIN-GH |
Department of Cell Biology and Institute of Biomembranes (P.v.K., G.J.S.), Experimental Cardiology Laboratory (M.S.), Interuniversity Cardiology Institute of the Netherlands (ICIN), University Medical Center Utrecht, Heidelberglaan 100, AZU-G02.525, 3584CX Utrecht, The Netherlands
Address all correspondence and requests for reprints to: Ger J. Strous, Ph.D., University Medical Center Utrecht, Department of Cell Biology, Heidelberg 100, AZU, G02.525, Utrecht 3584 CX, The Netherlands. e-mail: . strous{at}med.uu.nl
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
GH promotes not only postnatal longitudinal growth in children, but functions throughout an individual’s life in protein, fat, and carbohydrate metabolism. GH secretion is pulsatile and regulated by two hypothalamic hormones: GHRH, which stimulates secretion, and somatostatin, which inhibits secretion (8). In the GH-deficient adult, the effects on body composition, fat accumulation, and decreased muscle mass are evident. The effectiveness of a peptide hormone depends equally on its presence in the circulation as well as the availability of receptors at the plasma membrane of target cells. The GHR is synthesized in the endoplasmic reticulum (ER), processed in the Golgi complex, and transported to the plasma membrane. Receptor turnover is rapid with a half-life of about 60 min, and hormone-accelerated receptor down-regulation has been observed (9, 10, 11). Two mechanisms are known to contribute to receptor turnover: Proteolysis at the cell surface, resulting in shedding of GH-binding protein (GHBP) (12) and ligand-induced endocytosis. Recently, it was shown that the metalloprotease, TNF
-converting enzyme, is involved in the shedding of GHBP (13) and that phorbol esters and growth factors can affect GHR proteolysis (14). In ligand-induced endocytosis, the receptor is also removed from the cell surface, an event that is followed by lysosomal degradation. It was shown that ongoing protein synthesis is important to maintain GH binding capacity (15).
The ubiquitin-proteasome pathway is involved in endosomal trafficking of membrane receptors, transporters and channels (reviewed in Refs. 16 and 17). Several mammalian receptor proteins, such as epidermal growth factor (EGF) receptor (18), platelet-derived growth factor receptor (19), c-kit receptor (20), T cell receptor (21), and the Met receptor (22) are ubiquitinated in response to ligand binding. The GHR was initially found to be ubiquitinated on amino acid sequencing of the receptor from rabbit liver (23). Ubiquitination at the plasma membrane usually targets proteins for degradation in the lysosome. The tyrosine kinase adaptor protein, c-Cbl, is an E3 ubiquitin ligase that mediates EGF receptor ubiquitination and promotes sorting of the receptor into multivesicular bodies, thereby attenuating kinase signaling (24). For the Met receptor, it was recently shown that degradation occurs mainly in lysosomes and that proteasome inhibitors interfere with Met receptor endocytic trafficking (25). Ligand-induced endocytosis of the GHR is mediated by the ubiquitin-proteasome pathway via a 10-amino acid motif within the cytoplasmic tail [ubiquitin-dependent endocytosis (UbE) motif; DSWVEFIELD] (26) and is inhibited in the presence of proteasome inhibitors (27). Most of the above-mentioned receptors are long-lived and are rapidly degraded in response to ligand binding and signal transduction, this process is often referred to as signal down-regulation (for reviews, see Refs. 28 and 29). In contrast, the GHR is short-lived and continuously degraded, whether or not GH is present (9, 30).
In this study, we investigate whether the ubiquitin-proteasome pathway is involved in the rapid turnover of the GHR, thereby controlling the availability of the receptor at the cell surface. By comparing the turnover of the GHR in the absence of ligand with the turnover of an endocytosis-deficient receptor lacking an active UbE motif, GHR(F327A), we show that the half-life of the mutant GHR is prolonged almost 3-fold. A GH antagonist, B2036, which can bind but not properly dimerize the receptors, due to a mutation in binding site 2 (G120K) (31), was internalized by the GHR and delivered to the lysosome for degradation. Internalization and degradation of B2036 was dependent on an active ubiquitin-proteasome pathway and could be inhibited using proteasome inhibitors. Using B2036 as an inactive receptor tag, we measured both GHR internalization and GHR-proteolysis, generating GHBP. Our data show that ubiquitin proteasome pathway-mediated internalization is the major determinant in GHR turnover. Thus, the ubiquitin-proteasome pathway controls the time-span of the GHR at the cell surface and its availability for signaling.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
supplemented with 10% FCS, 4.5 g/l glucose, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.45 mg/ml geneticin. Previously, it was shown that sodium butyrate can enhance expression levels of proteins under the control of the cytomegalovirus early promoter (35). For experiments, cells were grown in 60-mm dishes in the absence of geneticin to a confluence of approximately 75% and 10 mM sodium butyrate was added overnight to increase GHR expression (33). Treatment of transfected cells with sodium butyrate did not alter the behavior of the GHR in any of the parameters examined in this study.
Antibodies and materials
Polyclonal antibodies to the GHR cytosolic tail were raised in rabbits against fusion proteins of glutathione-S-transferase and GHR peptides consisting of amino acids 271–318 (anti-T) or 327–493 (anti-B) as described before (27). The antibody mAb5 recognizing the lumenal part of the GHR was from AGEN Inc. (Parsippany, NJ). The antiserum against human GH (hGH) was raised in rabbits (36). The proteasome inhibitor MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal) was from Calbiochem. hGH was a gift from Lilly Research Labs (Indianapolis, IN). The GH antagonist B2036 is mutated at binding site 2 with G120K. In addition to the G120K mutation, there are 8 additional substitutions that enhance binding site 1 affinity (31). B2036 was kindly supplied by William F. Bennett of Sensus Drug Development Corp. (Austin, TX).
Metabolic labeling
Cells were grown in 60-mm dishes and incubated in methionine- and cysteine-free MEM, [35S]methionine (3.7 megabecquerels/ml Tran (35)S Label, 40 terabecquerels/mmol, ICN Biochemicals, Costa Mesa, CA) was added and the incubation was continued for 10 min at 30 C in a CO2-incubator. The radioactivity was replaced with medium containing 100 µM unlabeled methionine, 0.1% BSA, and chased for 5–240 min. Cells were lysed and samples were immunoprecipitated (see below). Radioactivity was determined using a Storm imaging system (Molecular Dynamics, Inc., Sunnyvale, CA) and quantified with Molecular Dynamics, Inc. ImageQuant software version 4.2a.
Ligand binding, internalization and degradation
125I-hGH and 125I-B2036 were prepared using chloramine-T resulting in a specific activity of 8 µCi/µg (37). For internalization and degradation studies, cells were grown in 12-well plates, washed with MEM supplemented with 20 mM HEPES (pH 7.4), and 0.1% BSA and incubated with 125I-GH (8 nM) or 125I-B2036 (8 nM) for 6 min at 30 C. The radioactivity was aspirated and the cells were washed and incubated in medium without ligand. At the indicated times, the medium was collected and precipitated with one volume of ice-cold 20% trichloroacetic acid (TCA) for 30 min on ice. TCA-soluble radioactivity was determined in the supernatant after centrifugation and was used as a measurement for degraded ligand. TCA-precipitated radioactivity was determined in the pellet and was used as a measurement for intact ligand. Membrane-associated ligand was removed by acid wash (0.15 M NaCl, 50 mM glycine, 0.1% BSA adjusted to pH 2.2 with 1 N HCl) for two times 5 min on ice. Internalized ligand was determined by measuring the radioactivity after solubilization of the acid treated cells in 1 N NaOH using the LKB Wallac Compugamma counter (Perkin-Elmer Lifesciences Inc., Boston, MA). Nonspecific radioactivity was determined in the presence of excess unlabeled ligand (9 µg/ml) and subtracted.
Gel filtration
Cells were grown in 60-mm dishes, washed with MEM supplemented with 20 mM HEPES (pH 7.4) and 0.1% BSA, and incubated with 125I-B2036 (8 nM) for 2 h on ice. Cells were washed free of unbound 125I-B2036, 1 ml of PBS-complete (PBS + 1 mM CaCl2 + 0.5 mM MgCl2) + 0.1% BSA was added and the incubation was continued for 30 min at 30 C. The medium was collected, centrifuged, and the supernatant analyzed on a HiLoad 16/60 Superdex 200 FPLC column (Amersham Pharmacia Biotech, Buckinghamshire, UK). Fractions (1.2 ml) were collected and counted in the LKB Compugamma counter.
Cross-linking
Cells were grown in 60-mm dishes, washed with MEM supplemented with 20 mM HEPES (pH 7.4) and 0.1% BSA and incubated with 125I-GH (8 nM) or 125I-B2036 (8 nM) for 2 h on ice. Cells were washed three times with PBS-complete on ice before 1 mM disuccinimidylsuberate (DSS; Perbio Science, Erembodegem-Aalst, Belgium) freshly dissolved in dimethylsulfoxide was added. After 30 min incubation with the cross-linker, cells were washed with MEM supplemented with 20 mM HEPES (pH 7.4) and 50 mM glycine to quench the unreacted DSS. Cells were lysed, and the supernatant was immunoprecipitated.
Proteinase K treatment
Cells were treated with proteinase K on ice to degrade the extracellular domain of the GHR and to demonstrate the effect of ligand binding on the degradation. Cells were grown in 60-mm dishes, washed with MEM supplemented with 20 mM HEPES (pH 7.4) and 0.1% BSA and incubated with GH (8 nM) or B2036 (8 nM) for 2 h on ice. Cells were washed three times with PBS on ice and 700 µl 0.5 mg/ml proteinase K (Roche Molecular Biochemicals, Mannheim, Germany) in PBS + 1 mM EDTA was added. Cells were incubated on ice for 30 min, and the detached cells were transferred to a test tube. Following centrifugation for 5 min at 300 x g at 4 C, the cell pellet was washed 3 times with 1 ml PBS + 2 mM phenylmethylsulfonyl fluoride and lysed in 300 µl lysis buffer. The lysate was centrifuged for 5 min at 14,000 x g and 50 µl of the supernatant was boiled with 50 µl of a 2x Laemlli SDS sample buffer containing 40 mM dithiothreitol.
Cell lysis, immunoprecipitation, and Western blotting
Cells were lysed on ice in 0.3 µl of lysis buffer containing 1% Triton X-100, 1 mM EDTA in PBS, with 50 mM NaF, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µM MG-132, and 1 mM phenylmethylsulfonyl fluoride. Immunoprecipitation of the supernatant was carried out in 1% Triton X-100, 0.5% SDS, 0.25% sodium deoxycholate, and 0.5% BSA in PBS with the various inhibitors. The lysates were incubated with the indicated antibodies for 2 h on ice and immune complexes were isolated using protein A-agarose beads (Repligen Co., Cambridge, MA). The immunoprecipitates were washed twice with the same buffer and twice with 10-fold diluted PBS. Immune complexes, isolated from equal aliquots of cell extracts, were subjected to SDS-PAGE using a precast ready gel system (Bio-Rad Laboratories, Inc., Veenendaal, The Netherlands) and transferred to polyvinylidenedifluoride paper as described (38). The blots were immunostained using the polyclonal GHR antibodies anti-T (1:2000 dilution) or anti-B (1:2000 dilution) or the monoclonal antibody mAb5 (1 µg/ml) followed by peroxidase-conjugated protein A (0.2 µg/ml) or rabbit-antimouse IgG (0.25 µg/ml) (Pierce Chemical Co.) and detected by enhanced chemiluminescence (Roche Molecular Biochemicals). To reprobe blots, the membranes were incubated for 1 h at room temperature in 0.15 M NaCl, 50 mM glycine (pH 2.5) buffer. The efficiency of the stripping procedure was checked and was found to remove greater than 95% of the signal. For quantification, preflashed X-OMAT UV films (Kodak, Rochester, NY) were used and scanned with a LKB Ultroscan XL Enhanced Laser Densitometer within the linear range of the film and the densitometer and analyzed using Gelscan XL version 2.1 (Amersham Pharmacia Biotech).
Microscopy
Cy3-GH and Cy3-B2036 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 (pH 7.4), 0.1% BSA and for 30 min with Cy3-GH (1 µg/ml) or Cy3-B2036 (1 µg/ml). Cells were washed with PBS to remove unbound label and fixed for 2 h in 3% paraformaldehyde in PBS. After fixation, the cells were embedded in Mowiol, and confocal laser scanning microscopy was performed using a Leica Corp. TCS 4D system.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). The GHR was synthesized as an 110-kDa glycoprotein precursor (p) and, with a half-life of about 55 min, converted to an 130-kDa mature receptor protein (m) due to complex glycosylation in the Golgi complex (Fig. 1C, left panel). The amount of mature protein was maximal between 60 and 120 min of chase and decreased rapidly thereafter, both in the absence and presence of GH. This result indicates that the receptor turnover rate is high regardless of whether or not it is occupied. At this stage, it is important to note that GH accelerated degradation of the receptor, slightly, but reproducibly. The half-life of the receptor, from synthesis to degradation, was estimated at 130 min (Fig. 1C, right panel). When we assume total conversion of the precursor to mature protein and consider the precursor half-life of 55 min, this implies a half-life of about 75 min for the mature protein. To determine the extent to which receptor shedding contributes to its turnover, we used the endocytosis-defective mutant receptor, GHR(F327A) (38, 39) (Fig. 1B). The residue Phe-327 is part of the UbE motif in the GHR cytosolic tail and essential for both ubiquitination and internalization of the GHR (26). Like the wild-type receptor, GHR(F327A) was synthesized as an 110-kDa precursor glycoprotein and converted to the 130-kDa mature species. Quantification of the radioactive precursor protein (Fig. 1C, left panel) showed nearly identical kinetics for its disappearance with a estimated half-life of 60 min. Maturation was also comparable, with 40% of the initial synthesized protein converted to the mature species after 60 min of chase. Degradation of the 130 kDa GHR(F327A) was clearly inhibited compared with the wild-type receptor, resulting in a delayed decrease in total radioactivity (precursor plus mature), with an estimated half-life of 240 min (Fig. 1C, right panel). Thus, when ubiquitin-proteasome pathway dependent endocytosis was impaired, the half-life of the mature receptor was prolonged 2.5 times (180 min). In the presence of GH, a slow migrating form of the receptor could be detected in the top of the gel (see arrow), which is more stable for GHR(F327A). Although the samples were resolved by SDS-PAGE under reducing conditions, this band most likely reflects a GH-dependent disulfide linked GHR dimer, as was reported in IM-9 cells (40). The high molecular weight receptor form shows a lower electrophoretic mobility as the 220K molecular weight marker, which is consistent with the expected size of a GHR dimer of approximately 260K. The half-life of this complex is similar to that of the 130K band, suggesting that indeed endocytosis mediates its disappearance. The results indicate that activation of the GHR by GH is not critical for the rapid receptor turnover and agree with observations that were obtained with a GHR mutant that failed to bind JAK2 (41).
|
). The individual bands were quantified as explained in Materials and Methods and the ratio of the mature 130-kDa protein (m) and the precursor (p) was calculated. For the wild-type receptor, the ratio m/p was 1.2 and for the GHR(F327A) the ratio was 2.7. A direct comparison of the ratios of mutant and wild-type receptor reveals that the half-life of the mature form of the mutant receptor is prolonged 2.2-fold. This comparison agrees with the factor 2.4 (180 vs. 75 min), calculated from the pulse chase experiment.
Endocytosis is a major factor in the turnover of the GHR
From the previous experiment, we concluded that activation of the GHR is not an important factor in modulating its turnover rate. Regulation of the turnover in the absence of ligand could be caused by internalization and subsequent lysosomal degradation of the nonactivated receptor and/or by shedding the extracellular domain of the receptor from the cell surface. To demonstrate internalization of an nonactivated receptor, we used the GH antagonist, B2036, which cannot dimerize the GHR due to a mutation in binding site 2 (G120K). B2036 has an affinity similar to native GH in intact cells (42). In our experiments, B2036 did not induce phosphorylation of the GHR, and was able to abolish GH-induced phosphorylation if added in a 5 times molar excess to GH. Furthermore, B2036 did not influence the maturation and half-life of the receptor (data not shown). To determine whether the wild-type GHR was able to internalize the antagonist and direct it to the degradative pathway, cells were incubated with either 125I-GH or 125I-B2036 and chased for various times in the absence of ligand. To measure both uptake and degradation, internalized radioactivity and TCA-soluble radioactivity were analyzed (Fig. 2). Both GH and B2036 were found intracellular after 15 min of chase, with maximum levels of about 40% after 30 min. Thereafter, the amount of intracellular ligand decreased with a concomitant increase of degraded ligand as TCA-soluble radioactivity in the medium. These results show that B2036 is internalized and degraded through the GHR, confirming that endocytosis and lysosomal degradation contribute to the rapid turnover of the nonactivated GHR.
|
, A and E), which confirms the internalization of the B2036, as shown in Fig. 2. Internalization of both Cy3-GH and Cy3-B2036 was completely inhibited following inactivation of the ubiquitin-proteasome pathway at the nonpermissive temperature (Fig. 3, B and F). Therefore, not only does ligand-induced endocytosis but also endocytosis of the nonactivated GHR depend on an active ubiquitin-proteasome pathway. Untransfected ts20 cells showed neither Cy3-B2036 binding nor internalization (not shown).
|
, C and G). The UbE motif is required for ligand-induced GHR internalization (26). Here, we show that mutating the UbE motif, as in GHR(F327A), results in a almost complete inhibition of uptake of both Cy3-GH and Cy3-B2036, indicating that internalization of the nonactivated receptor requires an intact UbE motif (Fig. 3, D and H). In addition, internalization studies that were performed with the use of 125I-GH and 125I-B2036 demonstrated both uptake and degradation in wild-type GHR expressing cells. Uptake and degradation were inhibited in cells incubated with MG-132 as well as in GHR(F327A) expressing cells (not shown). These results demonstrate that, besides ligand-induced GHR endocytosis, endocytosis of the nonactivated GHR depends on the ubiquitin-proteasome pathway.
GHBP shedding contributes little to the availability of the GHR
Receptor proteolysis by TNF-converting enzyme results in shedding of GHBP and constitutes the third mechanism which may regulate the availability of the GHR at the cell surface. GHBP is the soluble extracellular domain of the GHR and is released, in rabbits, as a 57-kDa protein (43). The exact cleavage site has not been established but is thought to be located at the membrane boundary (23). Using 125I-B2036, we noticed a considerable amount of TCA-precipitable radioactivity in the medium, which was not observed with GH (Fig. 4A). To further characterize the nature of this radioactivity, the medium was analyzed on a gel filtration column and compared with iodinated antagonist which was not incubated with cells. Figure 4B shows that the majority of the radioactivity, released from the cells, eluted as a complex that was bigger in size than 125I-B2036. To characterize the complex, the medium from B2036-treated cells was immunoprecipitated with anti-GH, an antibody which also recognizes the GH-antagonist. As seen in Fig. 4C, a protein could be detected after immunoprecipitation which reacted with an antibody directed against the extracellular domain of the GHR (mAb5) but not with an antibody against the cytosolic tail (anti-T). The size of the detected protein is the same size as would be expected for the GHBP (57 kDa), indicating that the extracellular domain of the GHR coimmunoprecipitated with B2036. In the cell lysates (cells), both antibodies detected the 110-kDa precursor (p) and the 130-kDa mature (m) forms of the receptor. Together, these results show that the release of TCA-precipitated B2036 into the medium is due to proteolysis of the GHR, which results in shedding of GHBP.
|
, after 30 min of chase, 13% of the 125I-B2036 was bound to GHBP in the medium pellet, whereas at the same time, 40% was detected intracellular (in Fig. 2). This result indicates that the internalization pathway is approximately three times more efficient in removing GHR from the cell surface than extracellular proteolysis. The cytosolic tail sequence does not play a role in GHBP release because truncated GH receptor isoforms with intracellular domains of only seven amino acids, lacking both boxes 1 and 2, produce large amounts of GHBP (44, 45). Therefore, it is unlikely that mutations in the UbE motif of the receptor tail affect GHBP shedding. To define the role of the UbE motif in both mechanisms, we compared the shedding process in the wild-type GHR and mutant GHR(F327A). Cells were incubated with 125I-GH or 125I-B2036, chased for 90 min in the absence of ligand, and TCA-precipitated radioactivity was measured in the medium (Fig. 4D). The endocytosis-deficient GHR(F327A) mutant showed an increased amount of precipitated 125I-B2036 (22%), compared with 13% in the wild-type GHR-expressing cells, while release of intact 125I-GH was low (6% and 4%, respectively). This result shows that the prolonged half-life of GHR(F327A) as measured in Fig. 1 is not due to an inhibition of receptor proteolysis. Instead GHBP release increased, a change that was most likely due to an inhibition of endocytosis. For truncated receptor isoforms, it is suggested that the lack of internalization of these receptors can cause increased amounts of GHBP, as more receptor is available for shedding (44). Inhibition of endocytosis with the proteasome inhibitor MG-132 also resulted in an increase in the release of 125I-B2036 (Fig. 4D). As the process of GHR shedding is probably not affected by the presence of B2036, as it is by GH (46), we conclude that conditions of complete inhibition of GHR endocytosis identify the process of shedding as an important factor for the half-life of the GHR at the cell surface. However, under standard conditions with no GH present, we conclude that internalization and subsequent lysosomal degradation is the main process in GHR turnover. Inhibition of endocytosis, although it increases GHR proteolysis, results in a 200% increase in receptors at the cell surface. Thus, for the greater part, the availability of the GHR at the cell surface of Chinese hamster lung cells is regulated by the rate of (ubiquitin-proteasome pathway-dependent) endocytosis, and for a minor part by the shedding process.
GH, not B2036, induces a conformational change in the GHR
As argued above, the GH antagonist B2036 is an useful tool to monitor the behavior of the nonactivated GHR. B2036 binds to the GHR without interfering with either its shedding, or with its endocytosis. Notably, GH-induced dimerization inhibits GHR proteolysis (46). This result raises the issue whether and by what means receptor dimerization changes the configuration of the GHR, and renders it inaccessible for metalloprotease-mediated proteolysis. To further validate our results with B2036, we examined two important parameters: 1) its behavior in dimerization experiments, and 2) its effect in a protease protection assay. Cells were cross-linked after binding of 125I-GH (G) or 125I-B2036 (A) to wild-type GHR-expressing cells, and the resulting complexes were isolated by immunoprecipitation with either anti-GH (not shown) or anti-GHR (Fig. 5A). Two radiolabeled protein complexes of the same electrophoretic mobility were detected by autoradiography for both 125I-GH and 125I-B2036. These complexes were neither observed in cells that do not express GHR, nor in the presence of excess amounts of unlabeled GH or B2036 (not shown and E in Fig. 5A). The lower molecular weight complexes migrated at a size of 150K, slightly above the mature receptor. Because they were immunoprecipitated with anti-GHR and contained either 125I-GH or 125I-B2036, they most likely represent GHR1:GH1 complexes. The complexes at the top of the gel resembled, according to their size, GHR-GH complexes with a stoichiometry of 2:1. Again, both GH- and B2036-containing complexes were of exactly the same size, and therefore, must be of the same stoichiometry. These results show that, despite its mutation at binding site 2, B2036 is able to form a heterotrimeric complex with two receptor subunits and imply that GH, but not B2036, induces a conformational change in the receptor complex thereby protecting the receptor against the sheddase.
|
). When no ligand was present (-), the mature GHR (m) at the cell surface was completely degraded by the protease, leaving only the 110-kDa ER species (p) intact. In addition, digestion of the GHR resulted in a cytosolic tail fragment of approximately 65 kDa (t), which was visible with the anti-tail antibody (anti-B), but not with the antibody against the extracellular domain (mAb5). This result clearly shows that, at steady state, all the mature GHR is present at the cell surface. When cells were incubated on ice with GH, before the proteinase K digestion, the mature form was partially protected against degradation and no formation of the cytosolic tail fragment could be observed. Incubation with B2036 did not protect the GHR against proteinase K digestion. The experiment was repeated with a 5-fold higher concentration of ligand and gave the same result. Our data show that GH, not B2036, induces a conformational change in the GHR2 GH1 complex, which protects it from digestion by proteinase K. Combined with the observation that GH also prevents shedding of GHBP (Fig. 4), our results show that the divalent GH, and not the monovalent B2036, shields the GHR/induces proteolytic protection. The results provide strong evidence for a dimerized status of the GHR, which is not protected against extracellular proteolysis in the absence of the activating ligand. Use of the monovalent GH B2036 is, therefore, a valid tool to monitor the behavior of the nonactivated GHR. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The availability of the GHR is determined by its residence-time at the plasma membrane. A vast majority of the mature form of the receptor was sensitive to proteinase K digestion, indicating that this form of the GHR resides at the cell surface (Fig. 5B). We used pulse chase labeling with [35S]methionine to calculate the half-lives of the precursor and mature receptor (Fig. 1C). As no ER-degradation of the GHR was detectable (33), complete conversion of the precursor into mature receptor protein was assumed. For IM-9 cells, it was reported that the GHR becomes progressively detergent insoluble in response to GH (47). By solubilizing cell pellets in SDS after cell lysis, no evidence was found that the GHR becomes insoluble in TX-100 in response to GH in Chinese hamster cells and, therefore, we assume that the loss of signal is due to receptor degradation. Based on these observations we calculated that the half-life of the mature, cell surface, form of the GHR is 75 min.
Which factors contribute to the rapid turnover of the cell surface receptor? One factor is endocytosis; inhibition of endocytosis results in a 2- to 3-fold prolonged half-life of the unoccupied GHR. Two independent methods were used to compare the half-lives of the mature form of the wild-type GHR with an endocytosis-deficient mutant. In the first method, we conducted pulse-chase labeling, and in the second method, we determined, at steady state, the ratio between the mature and precursor protein. We were permitted to compare the ratio mature/precursor protein because the pulse chase labeling showed that the exit rate from the ER and maturation in the Golgi complex was identical for both receptors. Moreover, also the mature form of the mutant receptor is completely proteinase K sensitive, and thus localized to the cell surface (not shown). Proteolytic shedding of GHBP is the second factor that influences the half-life of the cell surface receptor, and can be blocked by metalloprotease inhibitors (48). Recent studies demonstrated that GH, but not the GH antagonist G120K, was able to inhibit phorbol ester-stimulated GHR proteolysis (46). We show that, by using B2036, it is possible to measure constitutive endocytosis and shedding at the same time. Our experiments revealed that endocytosis contributed three times more to turnover of the cell surface receptor than shedding in wild-type GHR expressing cells. If endocytosis and shedding are the only factors determining the turnover of the GHR, then inhibition of shedding in the endocytosis-deficient mutant should result in an extended half-life of the mature receptor. Incubation with GH results in inhibition of shedding, but we still observe some degradation of the GHR(F327A) in the presence of GH (Fig. 1B), indicating that there might be a third factor involved in the degradation. Precise quantification of this factor is difficult without knowing the type of degradation that may be involved. However, the fact that inhibition of endocytosis [in GHR(F327A)], together with inhibition of shedding (GH), results in a 3- to 4-fold increase in half-life of the cell surface GHR, indicates that these two processes contribute significantly to receptor degradation. A low amount of degradation that occurs in the GHR(F327A) mutant expressing cells after inhibition of shedding may be due to ubiquitin-proteasome pathway independent endocytosis, which was previously reported in these cells (27).
Because we use transfected cell lines that overexpress the GHR, it was important to establish that the number of GHRs per cell does not affect any of the parameters determining availability. Previously, we showed that increasing concentrations of sodium butyrate (0–10 mM) increase the GHR expression in CMV promoter containing pcDNA3.1 plasmids up to 5-fold without affecting the ratio mature/precursor of the GHR (33, 35). This proves that the half-life of the cell surface receptor remains the same over a considerable range of receptor numbers. The constitutive turnover of the GHR is probably cell type-dependent and is fast in most cell types: in rat adipocytes (T
= 45 min) (9), in rat liver (T = 40 min) (30), and in mouse fibroblasts (T = 75 min) (10). GH, but not the nondimerizing mutant G120R, induces rapid down-regulation of cell surface receptors in IM-9 lymphocytes (49). In these cells, GH incubation results in irreversible binding and accumulation of long-lived, detergent-insoluble receptors at the cell surface (40, 47). Because this down-regulation does not necessarily result in receptor degradation, IM-9 cells may contain a different balance between the factors contributing to GHR down-regulation (50). Our data indicate that, in Chinese hamster ts20 cells, degradation is mainly determined by the ubiquitin-proteasome pathway-dependent internalization. Future experiments, in which the activity of the ubiquitin pathway can be varied, will provide definitive proof. With the antagonist as a tool to demonstrate receptor internalization and shedding, it will be possible to determine the contribution of both mechanisms in different cell types and various conditions.
The current data support a model in which GH induces a conformational change of the GHR, thereby inhibiting the release of GHBP. Earlier studies have suggested that such a conformational change is required for signal transduction (51, 52). The fact that GH was able to protect the GHR from digestion by proteinase K, while B2036 did not, supports this model. B2036 was cross-linked to the GHR in a high molecular weight complex which suggests that the GHR can exist as a preformed dimer at the cell surface, as was shown for the erythropoietin receptor (53, 54, 55). In this model, the site 2 domain in GH would induce a conformational change in the complex resulting in receptor activation, rather than recruiting the second receptor. Recently, Ross and co-workers (56) also provided evidence for binding of B2036 to a receptor dimer. The existence of preformed dimers might be beneficial for efficient signaling at low receptor densities. The majority of the complexes we detected after cross-linking represented, as judged from size, however a GH1GHR1 complex. We do not know at this point whether this is due to inefficient cross-linking or to a equilibrium at the cell surface between preformed dimers and real monomeric receptors.
Regulation of receptor number at steady state is of prime importance for GH signal transduction. Here, we provide strong evidence that the ubiquitin-proteasome pathway controls the number of cell surface GH receptors by controlling its constitutive internalization. Supraphysiological levels of glucocorticoids, whether endogenous (Cushing’s syndrome) or exogenous (glucocorticoid therapy), inhibit growth in children. Glucocorticoids activate the ubiquitin-proteasome pathway, and the artificial glucocorticoid, dexamethasone, antagonizes cellular GH action by decreasing GH binding (57, 58), indicating an inversed relation between the activity of the ubiquitin proteasome pathway and GH binding. In this respect, it is interesting to note that the ubiquitin-proteasome pathway is likely the universal system for the degradation of muscle protein induced by starvation (59), sepsis (60), metabolic acidosis (61), denervation atrophy (62), burns (63), and diabetes mellitus (64). In patients with cancer that undergo a loss of skeletal muscle mass, a condition known as cachexia, the increase in protein breakdown is thought to be associated with an up-regulation of the ubiquitin-proteasome pathway (65). The characteristic features of critical illness, like increased protein turnover and a negative nitrogen balance, are partly attributable to resistance to GH (66). From our results, it is expected that up-regulation of the ubiquitin-proteasome pathway results in the down-regulation of the number of GHRs at the cell surface, thereby providing an possible explanation for the decreased protein synthesis in these patients.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Abbreviations: DSS, Disuccinimidylsuberate; EGF, epidermal growth factor; ER, endoplasmic reticulum; GHBP, GH binding protein; GHR, GH receptor; hGH, human GH; IRS, insulin receptor substrate; JAK, Janus kinase; TCA, trichloroacetic acid; UbE, ubiquitin-dependent endocytosis.
Received October 3, 2001.
Accepted for publication December 26, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
converting enzyme (TACE) is a growth hormone binding protein (GHBP) sheddase: the metalloprotease TACE/ADAM-17 is critical for (PMA-induced) GH receptor proteolysis and GHBP generation. Endocrinology 141:4342–4348This article has been cited by other articles:
 |
|
 |
|
  C. A. Oliveira, A. B. Victor-Costa, and R. A. Hess Cellular and Regional Distributions of Ubiquitin-Proteasome and Endocytotic Pathway Components in the Epithelium of Rat Efferent Ductules and Initial Segment of the Epididymis J Androl, September 1, 2009; 30(5): 590 - 601. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Kenth, J. A M. Mergelas, and C. G. Goodyer Developmental changes in the human GH receptor and its signal transduction pathways J. Endocrinol., July 1, 2008; 198(1): 71 - 82. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. van Kerkhof, J. Putters, and G. J. Strous The Ubiquitin Ligase SCF(betaTrCP) Regulates the Degradation of the Growth Hormone Receptor J. Biol. Chem., July 13, 2007; 282(28): 20475 - 20483. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Deng, K. He, X. Wang, N. Yang, C. Thangavel, J. Jiang, S. Y. Fuchs, and S. J. Frank Determinants of Growth Hormone Receptor Down-Regulation Mol. Endocrinol., July 1, 2007; 21(7): 1537 - 1551. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. M. van den Eijnden, L. L. Lahaye, and G. J. Strous Disulfide bonds determine growth hormone receptor folding, dimerisation and ligand binding J. Cell Sci., August 1, 2006; 119(15): 3078 - 3086. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. N. Bullock, J. E. Debreczeni, A. M. Edwards, M. Sundstrom, and S. Knapp Crystal structure of the SOCS2-elongin C-elongin B complex defines a prototypical SOCS box ubiquitin ligase PNAS, May 16, 2006; 103(20): 7637 - 7642. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M J Waters, H N Hoang, D P Fairlie, R A Pelekanos, and R J Brown New insights into growth hormone action J. Mol. Endocrinol., February 1, 2006; 36(1): 1 - 7. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Landsman and D. J. Waxman Role of the Cytokine-induced SH2 Domain-containing Protein CIS in Growth Hormone Receptor Internalization J. Biol. Chem., November 11, 2005; 280(45): 37471 - 37480. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. He, K. Loesch, J. W. Cowan, X. Li, L. Deng, X. Wang, J. Jiang, and S. J. Frank Janus Kinase 2 Enhances the Stability of the Mature Growth Hormone Receptor Endocrinology, November 1, 2005; 146(11): 4755 - 4765. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. Cowan, X. Wang, R. Guan, K. He, J. Jiang, G. Baumann, R. A. Black, M. S. Wolfe, and S. J. Frank Growth Hormone Receptor Is a Target for Presenilin-dependent {gamma}-Secretase Cleavage J. Biol. Chem., May 13, 2005; 280(19): 19331 - 19342. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zhou, W. K. W. Ko, W. K. K. Ho, S. S. Stojilkovic, and A. O. L. Wong Novel Aspects of Growth Hormone (GH) Autoregulation: GH-Induced GH Gene Expression in Grass Carp Pituitary Cells through Autocrine/Paracrine Mechanisms Endocrinology, October 1, 2004; 145(10): 4615 - 4628. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Gent, M. van den Eijnden, P. van Kerkhof, and G. J. Strous Dimerization and Signal Transduction of the Growth Hormone Receptor Mol. Endocrinol., May 1, 2003; 17(5): 967 - 975. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Wang, K. He, M. Gerhart, Y. Huang, J. Jiang, R. J. Paxton, S. Yang, C. Lu, R. K. Menon, R. A. Black, et al. Metalloprotease-mediated GH Receptor Proteolysis and GHBP Shedding. DETERMINATION OF EXTRACELLULAR DOMAIN STEM REGION CLEAVAGE SITE J. Biol. Chem., December 20, 2002; 277(52): 50510 - 50519. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Gent, P. van Kerkhof, M. Roza, G. Bu, and G. J. Strous Ligand-independent growth hormone receptor dimerization occurs in the endoplasmic reticulum and is required for ubiquitin system-dependent endocytosis PNAS, July 23, 2002; 99(15): 9858 - 9863. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
, Wild-type GHR; 
, GHR(F327A). The result of one representative experiment is presented. D, Duplicate samples of wild-type GHR and GHR(F327A) expressing ts20 cells were lysed, separated by SDS-PAGE and immunoblotted using anti-GHR (mAb5). The individual bands were quantified using a laser equipped densitometer and the amount of mature GHR (m) was expressed compared with the amount of precursor protein (p).
65 kDa). Relative molecular weight standards (Mr x10-3) are shown to the right.