©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Degradation Process of Ligand-stimulated Platelet-derived Growth Factor -Receptor Involves Ubiquitin-Proteasome Proteolytic Pathway (*)

(Received for publication, July 18, 1995; and in revised form, September 12, 1995)

Seijiro Mori (1)(§) Keiji Tanaka (2) Satoshi Omura (3) Yasushi Saito (1)

From the  (1)Second Department of Internal Medicine, Chiba University School of Medicine, 1-8-1 Inohana, Chiba 260, Japan, (2)Institute for Enzyme Research, The University of Tokushima, Kuramoto-cho, Tokushima 770, Japan, and (3)Research Center for Biological Function, The Kitasato Institute, Minato-ku, Tokyo 108, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The platelet-derived growth factor beta-receptor undergoes polyubiquitination as a consequence of ligand binding. We have previously reported that ligand-induced ubiquitination of the receptor plays a negative regulatory role in its mitogenic signaling possibly by promoting the efficient degradation of the ligand-activated receptor (Mori, S., Heldin, C.-H., and Claesson-Welsh, L.(1993) J. Biol. Chem. 268, 577-583). In the present study, we have examined effects of different kinds of cell-penetrating proteasome inhibitors, including substrate-related peptidyl aldehydes, Cbz-Ile-Glu(O-t-Bu)-Ala-leucinal (where Bu is butyl and Cbz is benzyloxycarbonyl) (PSI) and Cbz-Leu-Leu-norvalinal (MG115), and a Streptomyces metabolite lactacystin, on degradation of the receptor in intact cells with the aim of evaluating the role of the receptor ubiquitination in the proteasome-dependent proteolytic process. These proteasome inhibitors were found to considerably inhibit ligand-stimulated degradation of the wild-type beta-receptor; however, their inhibitory effect was not observed when the cells expressing the ubiquitination-deficient mutant beta-receptor were analyzed. These data suggest that the degradation process of the ligand-stimulated beta-receptor involves the ubiquitin-proteasome proteolytic pathway.


INTRODUCTION

Most receptors for polypeptide growth factors have a similar overall structural organization with an extracellular ligand-binding domain, a single transmembrane-spanning region, and an intracellular ligand-stimulatable tyrosine kinase domain. Based on the degree of structural similarities, the receptor tyrosine kinases can be divided into subfamilies(1) . Binding of the growth factor activates the intrinsic tyrosine kinase activity of the receptor, which leads to receptor autophosphorylation and to phosphorylation of intracellular substrates (for reviews, see (2) and (3) ). One role for receptor autophosphorylation is to present binding sites for signal transduction molecules containing one or two copies of a so-called Src homology 2 domain, which mediates the interaction with the receptor(4, 5) . Ligand binding is also accompanied by clustering of the receptors, and the receptor-ligand complex is ultimately delivered to and degraded in lysosomes.

Platelet-derived growth factor (PDGF) (^1)promotes the growth of mesenchymal cells in normal and pathological processes(6) . Two types of the receptor for PDGF, designated alpha- and beta-receptors, have been identified(7, 8, 9) , and they both belong to the receptor tyrosine kinase subfamily III(10) . We have previously reported that the PDGF beta-receptor undergoes polyubiquitination as a consequence of ligand binding (11) and have suggested that the ligand-induced ubiquitination plays a negative regulatory role in mitogenic signaling of the PDGF beta-receptor, possibly by promoting the efficient degradation of the ligand-activated receptor(12) . Ubiquitin is present in eukaryotes and is a highly conserved 76-amino acid residue protein(13) . Evidence supports the concept that ubiquitin conjugation to protein is implicated in ATP-dependent proteolytic pathways for short-lived proteins such as cyclins, Myc, Fos, and p53 (see (14) for a review). A multisubunit 26 S (>2000 kDa) protease complex, which specifically degrades proteins conjugated to ubiquitin, has previously been described, and 20 S (750 kDa) proteasome, also commonly known as macropain or the multicatalytic proteinase complex, has subsequently been shown to be the proteolytic core of the 26 S complex (for reviews, see (15) and (16) ). The coupling of ubiquitin to proteins is catalyzed by a family of small carrier proteins called E2s with or without the participation of the ubiquitin-protein ligase, E3 (for reviews, see (17) and (18) ).

Recently, reagents that inhibit the ubiquitin-proteasome proteolytic pathway in intact cells have become available, including substrate-related peptidyl aldehydes (19, 20, 21) and a Streptomyces metabolite lactacystin(22) . In the present study, we report that these proteasome inhibitors also considerably inhibit ligand-stimulated degradation of the wild-type PDGF beta-receptor in vivo, and their inhibitory effect is lost when an ubiquitination-deficient mutant beta-receptor is analyzed, suggesting that the degradation process of the ligand-stimulated PDGF beta-receptor involves the ubiquitin-proteasome proteolytic pathway.


EXPERIMENTAL PROCEDURES

Chemicals

E64-D (2S,3S-t-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester) and chloroquine were purchased from Sigma. MG115 (N-benzyloxycarbonyl-L-leucyl-L-leucyl-L-norvalinal) and PSI (N-benzyloxycarbonyl-L-isoleucyl--t-butyl-L-glutamyl-L-alanyl-L-leucinal) were provided by Peptide Institute Inc. (Osaka, Japan). Calpeptin (N-benzyloxycarbonyl-L-leucyl-L-norleucinal) was provided by T. Tsujinaka(23) . Lactacystin was prepared as described(24, 25) . These drugs were dissolved in Me(2)SO before use and, throughout the experiments, the final concentration of Me(2)SO in cell culture media was kept 0.5% including control cultures.

Cells

Porcine aortic endothelial (PAE) cells expressing the wild-type human PDGF beta-receptor or a mutant PDGF beta-receptor where the carboxyl-terminal 98 amino acid residues were truncated (CT98 mutant) were prepared as described (26) and were cultured in Ham's F-12 medium (Life Technologies, Inc.) containing 10% fetal bovine serum (Life Technologies, Inc.) and 200 µg/ml of the antibiotic G418 (Life Technologies, Inc.).

Antisera

The rabbit peptide antiserum PDGFR-3 was generated using a synthetic peptide corresponding to the murine PDGF beta-receptor amino acids 981-994(7) ; this antiserum reacts in a specific manner with the human PDGF beta-receptor(27) . The rabbit peptide antiserum PDGFR-HL2 was provided by S. M. Weima(28) ; this antiserum was generated using a synthetic peptide corresponding to the murine PDGF beta-receptor amino acids 701-732(7) .

Ligands

Recombinant human PDGF-BB was purchased from R & D Systems (Minneapolis, MN). I-PDGF-BB (>1000 Ci/mmol) was from Amersham Corp.

I-PDGF-BB Binding Experiments

The experiments were performed essentially as described by Mori et al.(29) . Confluent cells in 12-well Costar dishes were incubated for 2 h at 37 °C with different drugs in 0.5 ml/well of a binding medium (Ham's F-12 medium containing 1 mg/ml bovine serum albumin). Then the cells were cooled down on ice and incubated in this medium with I-PDGF-BB (100,000 cpm/well) for 1 h at 4 °C. After washing, the cells were further incubated at 37 °C in the presence of the respective drugs for different time periods. The incubation medium was then removed and precipitated with an equal volume of 10% trichloroacetic acid. The amount of trichloroacetic acid-nonprecipitable radioactivity was taken as an estimate of ligand degradation. After removal of the medium, the cells were incubated for 5 min on ice with phosphate-buffered saline containing 1 mg/ml bovine serum albumin or, alternatively, with the same buffer adjusted to pH 3.7 with acetic acid. This acid wash procedure releases more than 90% of the cell surface-bound ligand into the medium (30, 31) and makes it possible to separately estimate the amount of internalized (acid-nonreleasable) ligand out of the total amount associated with the cells. After the acid wash, the cells were lysed in 1 N KOH to determine the amount of total and internalized cell-associated radioactivities.

Metabolic Labeling, Immunoprecipitation, and SDS-Polyacrylamide Gel Electrophoresis

Confluent 25-cm^2 flasks of cells were labeled in methionine- and cysteine-free modified Eagle's medium Select-Amine (Life Technologies, Inc.) supplemented with 100 µCi/ml EXPRESS [S]protein labeling mix (specific activity, >1000 Ci/mmol, DuPont NEN) for 2 h at 37 °C. Then the cells were incubated for 2 h at 37 °C with complete medium containing different drugs as well as a 10-fold molar excess of unlabeled methionine and cysteine to chase labeled receptors to the cell surface. Thereafter, the cells were incubated in the binding medium (see above) with 100 ng/ml PDGF-BB at 37 °C in the presence of the respective drugs for different time periods. After incubation, the cells were washed, lysed in a lysis buffer consisting of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 10 mM EDTA, 1% aprotinin (Sigma), 1 mM phenylmethylsulfonyl fluoride (Sigma), and 500 µM sodium orthovanadate and prepared for immunoprecipitation essentially as described by Mori et al.(12) . Immunoprecipitations were performed as described(12) , using the PDGFR-3 or PDGFR-HL2 antisera followed by adsorption to protein A-Sepharose CL-4B (Pharmacia Biotech Inc.). SDS-polyacrylamide gel electrophoresis was carried out in slab gels of 8% polyacrylamide (ProRanger, AT Biochem) according to Blobel and Dobberstein(32) . Gels were prepared for fluorography by soaking in Amplify (Amersham Corp.) and then dried and exposed to Hyperfilm MP (Amersham Corp.). Quantitation of the signal on the gel was performed using a PhosphorImager with the ImageQuant software (Molecular Dynamics).

Each experiment presented in this study was repeated at least twice under identical conditions to confirm the reproducibility of the observations.


RESULTS

Ligand-stimulated degradation of the PDGF beta-receptor is thought to play an important role in regulation of signal transduction by the receptor. Therefore, it was of interest to clarify the mechanism of the degradation process by analyzing effects of different protease inhibitors.

First, we used substrate-related peptidyl aldehydes, MG115 (19) and PSI (20) , as proteasome inhibitors and E64-D as a calpain inhibitor and examined their effects on ligand-stimulated degradation of the PDGF beta-receptor. PAE cells expressing the wild-type PDGF beta-receptor were metabolically labeled for 2 h and then chased for 2 h with or without the different drugs. Thereafter, the cells were stimulated with PDGF-BB for 0-120 min at 37 °C in the presence of the respective drugs. After incubation, the cells were lysed and immunoprecipitated with the beta-receptor-specific antiserum PDGFR-3, and the immunoprecipitates were analyzed by SDS-gel electrophoresis followed by fluorography. As shown in Fig. 1, panel A, before stimulation of the cells with PDGF-BB, the mature receptor band of 190 kDa was clearly detected in each lane (lanes 1, 6, 11, and 16). After PDGF-BB stimulation, the intensity of the band decreased rapidly in control (lanes 2-5) and E64-D-treated (lanes 7-10) cells, whereas the intensity decreased slowly in MG115- (lanes 12-15) and PSI-treated (lanes 17-20) cells. The efficiency of ligand-stimulated degradation of the receptor was assessed by the rate of decrease in intensity of the mature receptor band after PDGF-BB stimulation. As shown in Fig. 1, panel B, quantitative analysis of the gel revealed that, in control cells, the intensity of the band decreased to 20% of the initial value after 30 min of PDGF-BB stimulation and further decreased to an undetectable level after 90 min. Treatment of the cells with E64-D did not affect the rate of the receptor degradation. On the other hand, both the treatment with MG115 and that with PSI similarly decreased the efficiency of the receptor degradation; the intensity was approximately 50% after 30 min and was still around 20% after 120 min. These results indicate that MG115- and PSI-sensitive but E64-D-insensitive proteases are involved in the degradation process of the ligand-stimulated PDGF beta-receptor.


Figure 1: Effects of different protease inhibitors on ligand-stimulated degradation of the wild-type PDGF beta-receptor. Panel A, PAE cells expressing the wild-type human PDGF beta-receptor were labeled for 2 h at 37 °C with 100 µCi/ml EXPRESS [S]protein labeling mix (DuPont NEN) and then incubated for 2 h at 37 °C with 0.5% Me(2)SO (Control) (lanes 1-5) or different protease inhibitors, 50 µM E64-D (lanes 6-10), 50 µM MG115 (lanes 11-15), and 50 µM PSI (lanes 16-20), in the presence of an excess of unlabeled methionine and cysteine. Thereafter, the cells were incubated with 100 ng/ml PDGF-BB at 37 °C in the presence of the respective drugs for the indicated time periods. After incubation, the cells were lysed, immunoprecipitated with PDGFR-3, and analyzed by SDS-gel electrophoresis and fluorography. The migration position of the mature form of the PDGF beta-receptor (Rec) is indicated by an arrowhead. The relative migration positions of molecular mass standards (myosin, 220 kDa; phosphorylase b, 97.4 kDa) run in parallel are also indicated. Panel B, quantitation of the mature form of the PDGF beta-receptor on the gel shown in panel A. Intensity of the mature receptor band in samples derived from the cells treated with different protease inhibitors, E64-D (open squares), MG115 (closed circles), and PSI (closed squares), and from control cells (open circles) was measured using the PhosphorImager with the ImageQuant software and is expressed as percent of that at time zero.



Next, we examined individual as well as synergistic effects of chloroquine and MG115 on ligand-stimulated degradation of the PDGF beta-receptor. The wild-type receptor-expressing cells were treated with these drugs one by one, or simultaneously, under the same conditions as those described in the previous experiment. As shown in Fig. 2, treatment of the cells with MG115 reproducibly decreased the efficiency of the receptor degradation. On the other hand, chloroquine treatment did not appreciably affect the rate of the receptor degradation. Furthermore, simultaneous treatment with chloroquine and MG115 did not enhance the inhibitory effect of MG115 at all. These results indicate that lysosomal proteolysis is not involved, at least in the initial degradation step of the ligand-stimulated PDGF beta-receptor.


Figure 2: Effects of MG115 and chloroquine on ligand-stimulated degradation of the wild-type PDGF beta-receptor. The wild-type receptor-expressing cells were labeled as described in the legend to Fig. 1and then chased for 2 h at 37 °C with 0.5% Me(2)SO (control), 50 µM MG115, 100 µM chloroquine or both 50 µM MG115 and 100 µM chloroquine. Thereafter, the cells were incubated with 100 ng/ml PDGF-BB at 37 °C in the presence of the respective drugs for the indicated time periods. After incubation, the cells were lysed, immunoprecipitated with PDGFR-3, and analyzed by SDS-gel electrophoresis and fluorography. Intensity of the mature receptor band in samples derived from the cells treated with chloroquine (open squares), MG115 (closed circles), or both MG115 and chloroquine (closed squares), and from control cells (open circles) was measured and is expressed as described in the legend to Fig. 1.



In order to confirm the participation of proteasomes in the degradation process of the ligand-stimulated PDGF beta-receptor, we used another kind of proteasome inhibitor, lactacystin, which is the most specific proteasome inhibitor available to date(22) . We also examined the effect of another peptidyl aldehyde, calpeptin, which is a specific calpain inhibitor(23) . The wild-type receptor-expressing cells were treated with these drugs under the same conditions as those described in the previous experiment. As shown in Fig. 3, treatment of the cells with lactacystin decreased the efficiency of the receptor degradation, as expected. On the other hand, calpeptin treatment did not affect the rate of the receptor degradation. These results, together with the previous results using MG115 and PSI, strongly support the interpretation that the proteasomedependent proteolytic pathway is involved in the degradation process of the ligand-stimulated PDGF beta-receptor.


Figure 3: Effects of calpeptin and lactacystin on ligand-stimulated degradation of the wild-type PDGF beta-receptor. The wild-type receptor-expressing cells were labeled as described in the legend to Fig. 1and then chased for 2 h at 37 °C with 0.5% Me(2)SO (control), 30 µM calpeptin, or 100 µM lactacystin. Thereafter, the cells were incubated with 100 ng/ml PDGF-BB at 37 °C in the presence of the respective drugs for the indicated time periods. After incubation, the cells were lysed, immunoprecipitated with PDGFR-3, and analyzed by SDS-gel electrophoresis and fluorography. Intensity of the mature receptor band in samples derived from the cells treated with lactacystin (closed circles) or calpeptin (closed squares) and from control cells (open circles) was measured and is expressed as described in the legend to Fig. 1.



The wild-type PDGF beta-receptor undergoes polyubiquitination as a consequence of ligand binding, and we have found that a mutant PDGF beta-receptor lacking the carboxyl-terminal 98 amino acid residues of the receptor (CT98 mutant) does not undergo this posttranslational modification(11) . In order to evaluate the role of the receptor ubiquitination on the proteasome-dependent degradation process, we examined the effect of lactacystin on ligand-stimulated degradation of the ubiquitination-deficient mutant receptor. PAE cells expressing the wild-type or CT98 mutant receptors were treated with lactacystin under the same conditions as those described in the previous experiment. As shown in Fig. 4, treatment of the wild-type receptor-expressing cells with lactacystin reproducibly decreased the efficiency of the receptor degradation. On the other hand, in the CT98 mutant receptor-expressing cells, lactacystin treatment did not appreciably affect the rate of the receptor degradation. Furthermore, even without lactacystin treatment, the rate of degradation of the CT98 mutant receptor was lower compared with that of the wild-type receptor, as reported previously(11) ; the efficiency of receptor degradation in the CT98 mutant receptor-expressing cells was nearly comparable with that observed in the lactacystin-treated wild-type receptor-expressing cells. These results indicate that the degradation process inhibitable by lactacystin is dependent on ligand-induced ubiquitination of the PDGF beta-receptor.


Figure 4: Effect of lactacystin on ligand-stimulated degradation of the wild-type and CT98 mutant PDGF beta-receptors. PAE cells expressing the wild-type or CT98 mutant PDGF beta-receptors were labeled as described in the legend to Fig. 1and then chased for 2 h at 37 °C with 0.5% Me(2)SO (control) or 100 µM lactacystin. Thereafter, the cells were incubated with 100 ng/ml PDGF-BB at 37 °C in the presence of the respective drugs for the indicated time periods. After incubation, the cells were lysed, immunoprecipitated with PDGFR-3 for the wild-type and with PDGFR-HL2 for the CT98 mutant receptors, and analyzed by SDS-gel electrophoresis and fluorography. Intensity of the mature receptor band in samples derived from the cells treated with lactacystin (closed circles for the wild-type and closed squares for the CT98 mutant receptors) and from control cells (open circles for the wild-type and open squares for the CT98 mutant receptors) was measured and is expressed as described in the legend to Fig. 1.



Ligand-induced endocytosis of the PDGF beta-receptor precedes its intracellular degradation, and, hence, the efficiency of internalization can affect that of degradation of the receptor. Thus, it was necessary to examine effects of the drugs on ligand-induced internalization of the receptor. The wild-type receptor-expressing cells were preincubated for 2 h with chloroquine, lactacystin, or calpeptin, then allowed to bind I-PDGF-BB for 1 h at 4 °C, washed, and further incubated with the respective drugs at 37 °C for different time periods. After incubation and subsequent acid wash at pH 3.7 to displace ligand bound to cell surface receptors, the total and acid-nonreleasable (internalized) cell-associated radioactivities were determined. Fig. 5shows the ratio between the internalized and total cell-associated ligand (expressed as percent radioactivity internalized). Internalization of receptor-bound ligand occurred rapidly during the first 10 min and further increased slowly for up to 30 min of incubation. Treatment of the cells with chloroquine, lactacystin, and calpeptin did not appreciably affect the internalization. These results clearly rule out the possibility that the observed inhibitory effect of lactacystin on the receptor degradation is due to a decrease in the internalization of the PDGF beta-receptor.


Figure 5: Effects of different drugs on internalization of I-PDGF-BB bound to PAE cells expressing the wild-type PDGF beta-receptor. The wild-type receptor-expressing cells were incubated for 2 h at 37 °C with 0.5% Me(2)SO (closed circles), 100 µM chloroquine (open circles), 100 µM lactacystin (closed squares), or 30 µM calpeptin (open squares). Thereafter, the cells were cooled down on ice, incubated for 1 h at 4 °C with I-PDGF-BB (100,000 cpm/well), washed, and then incubated at 37 °C for the indicated time periods in the continuous presence of the respective drugs. The incubation was terminated by removal of the medium, and the cells were incubated for 5 min on ice with phosphate-buffered saline containing 1 mg/ml bovine serum albumin or, alternatively, with the same buffer adjusted to pH 3.7 with acetic acid (acid wash procedure). After the acid wash, the cells were lysed to determine the amount of acid-nonreleasable (internalized) and total cell-associated radioactivities. The rate of internalization is expressed as the ratio between the internalized and total cell-associated ligand (expressed as percent radioactivity internalized). The standard deviation at each point was less than 5% (triplicate determinations).



Finally, we also examined effects of chloroquine, lactacystin, and calpeptin on degradation of the receptor-bound ligand. The wild-type receptor-expressing cells were treated with these drugs under the same conditions as those described in the previous experiment. After incubation, the amount of trichloroacetic acid-nonprecipitable radioactivity in the medium, as a measure of degraded ligand, was recorded. Fig. 6shows the ratio between the degraded ligand and the initial cell-associated ligand (expressed as percent radioactivity degraded). Degradation of receptor-bound ligand increased rapidly in control cells, whereas the degradation increased somewhat slowly in lactacystin-treated cells. In calpeptin- and chloroquine-treated cells, appreciable degradation did not occur during 120 min of incubation. These results indicate that the degradation process of receptor-bound ligand is dependent completely on a chloroquine- and calpeptin-sensitive pathway as well as partially on a lactacystin-sensitive pathway.


Figure 6: Effects of different drugs on degradation of I-PDGF-BB bound to PAE cells expressing the wild-type PDGF beta-receptor. The wild-type receptor-expressing cells were treated with 0.5% Me(2)SO (closed circles), 100 µM chloroquine (open circles), 100 µM lactacystin (closed squares) or 30 µM calpeptin (open squares), and incubated with I-PDGF-BB under the same conditions as those described in the legend to Fig. 5. After incubation, the medium was subjected to trichloroacetic acid precipitation. The amount of trichloroacetic acid-nonprecipitable radioactivity was taken as a measure of degradation of I-PDGF-BB. After removal of the medium, the cells were lysed to determine cell-associated radioactivity. The rate of degradation is expressed as the ratio between the degraded ligand and the initial cell-associated ligand (expressed as percent radioactivity degraded). The standard deviation at each point was less than 6% (triplicate determinations).




DISCUSSION

Our present study demonstrates that, among different protease inhibitors examined, including MG115, PSI, and lactacystin as proteasome inhibitors, E64-D and calpeptin as calpain inhibitors, and chloroquine as an inhibitor for lysosomal proteolysis, only the proteasome inhibitors decrease the efficiency of ligand-stimulated degradation of the wild-type PDGF beta-receptor. Furthermore, the degradation process inhibitable by lactacystin is dependent on ligand-induced ubiquitination of the receptor; the inhibitory effect of lactacystin is lost when the ubiquitination-deficient mutant receptor (CT98 mutant) is analyzed. These data indicate that the ligand-induced ubiquitination and subsequent proteasome-dependent proteolysis are involved in the degradation pathway of ligand-stimulated PDGF beta-receptor.

The proteasome inhibitors used in the present study could not completely block the receptor degradation. One possibility that the dosage of the drugs was not enough to exert their full effects is unlikely, since 200 µM MG115 gave the same result (data not shown), and, more importantly, the rate of degradation of the wild-type receptor in lactacystin-treated cells was nearly comparable with that of the ubiquitination-deficient mutant receptor (CT98 mutant) (Fig. 4); the ubiquitin-proteasome proteolytic process must not occur in these conditions. Another possibility is that the degradation process of ligand-stimulated PDGF beta-receptor is catalyzed also by some distinct protease(s), which is resistant not only to the proteasome inhibitors but to the calpain inhibitors and chloroquine. However, a more plausible explanation is that a fraction of accumulated receptors after ligand-induced endocytosis might have become less soluble, e.g. through association with the detergent-insoluble cell fraction, which in our procedure was removed by centrifugation from the lysate before immunoprecipitation (33) .

Our present observation that chloroquine treatment did not affect the rate of the receptor degradation (Fig. 2) does not necessarily exclude the possibility that lysosomal proteolysis also contributes to the degradation of ligand-stimulated PDGF beta-receptor. Because our current method records the fate of the intact mature receptor molecule, that is to say the initial degradation step of the receptor, partially cleaved receptor molecules, if any, cannot be detected due to unpredictable changes in their molecular size or immunological reactivity. It is rather conceivable that, following the proteasome-dependent degradation of the receptor, the resultant peptide fragments are delivered to and further degraded in lysosomes.

As shown in Fig. 6, the degradation of receptor-bound PDGF-BB was completely inhibited by chloroquine, as expected. The result fits the notion that the ligand-receptor complex is ultimately delivered to and degraded in lysosomes. Calpeptin was also found to completely block the ligand degradation, apparently suggesting the involvement of calpains in the degradation process. However, at present we attribute the effect of calpeptin to its possible inhibition of lysosomal cysteine proteases. On the other hand, lactacystin partially inhibited the ligand degradation. The result raises a possibility that the ubiquitin-proteasome proteolytic process functions also for the degradation of receptor-bound ligand upstream of the lysosomal pathway. The interpretation is further supported by our previous observation that the degradation of receptor-bound PDGF-BB by the cells expressing the ubiquitination-deficient mutant receptor (CT98 mutant) was about half that by the wild-type receptor-expressing cells(12) .

Taken together, our current hypothesis concerning the degradation processes of the receptor-ligand complex of the PDGF beta-receptor is as follows (see Fig. 7). After ligand stimulation, the receptor is polyubiquitinated, and the internalized receptor-ligand complex is initially degraded by the ubiquitin-proteasome proteolytic machinery. Then the resultant peptide fragments are delivered to and further degraded in lysosomes. The functional association of the two, apparently distinct, proteolytic systems, the ubiquitin system and the lysosomal autophagic system, has been described for the heat-induced accelerated degradation of long lived proteins in the ts85 and the ts20 cells (which harbor a mutated thermolabile ubiquitin-activating enzyme, E1 (see (34) and (35) )). However, the cooperative proteolysis by proteasome and lysosome, as suggested by the present study, has not previously been reported. Further study is necessary.


Figure 7: Schematic illustration of possible modes of ligand-stimulated degradation of the PDGF beta-receptor. Ligand-activated receptor is polyubiquitinated, and then the receptor-ligand complex is internalized and initially degraded by the proteasome-dependent pathway. The resultant peptide fragments are delivered to and further degraded in lysosomes. Ub stands for ubiquitin conjugation.



In addition to the PDGF beta-receptor, other monomeric receptors belonging to different kinds of the receptor tyrosine kinase subfamily, such as the PDGF alpha-receptor, the epidermal growth factor receptor, the colony-stimulating factor-1 receptor, the fibroblast growth factor receptor-1(36) , and the c-kit-encoded protein receptor (37) , have recently been found to be polyubiquitinated after ligand stimulation. It is thus possible that the novel mechanism for down-regulation of signal transduction by the receptor, the ligand-induced receptor ubiquitination and its subsequent proteosomal degradation, reported in the present study for the PDGF beta-receptor is a general mechanism employed by most of the monomeric receptor tyrosine kinases. Our future studies will be aimed at exploring the possibility.


FOOTNOTES

*
This work was supported by Grants 07557222 and 07671104 from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-43-226-2089; Fax: 81-43-226-2095.

(^1)
The abbreviations used are: PDGF, platelet-derived growth factor; PAE, porcine aortic endothelial.


ACKNOWLEDGEMENTS

We are grateful to Dr. T. Tsujinaka (The Second Department of Surgery, Osaka University Medical School, Japan) for calpeptin and to Dr. S. M. Weima (The Hubrecht Laboratory, Netherlands Institute for Developmental Biology, The Netherlands) for the PDGFR-HL2 antiserum.


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