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Receptor-Associated Protein Binding Blocks Ubiquitinylation of the Low Density Lipoprotein Receptor-Related Protein
Archives of Biochemistry and Biophysics Vol. 396, No. 1, December 1, pp. 106 –110, 2001 doi:10.1006/abbi.2001.2597, available online at http://www.idealibrary.com on
Receptor-Associated Protein Binding Blocks Ubiquitinylation of the Low Density Lipoprotein Receptor-Related Protein U. K. Misra and S. V. Pizzo 1 Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710
Received August 16, 2001
The low density lipoprotein receptor-related protein (LRP) consists of two subunits, M r ⬃ 515,000 and 85,000. LRP is a receptor for activated ␣ 2-macrogobulin (␣ 2M*), Pseudomonas exotoxin A, and many other proteins. We now report that ubiquitinylation of the LRP heavy chain occurred when either Pseudomonas exotoxin A or ␣ 2M* bound to LRP on macrophages. Ubiquitinylation was dose-dependent and maximal about 30 min after ligation of the receptor. Addition of the proteosome inhibitor MG-132 sustained the level of ubiquitin-LRP for longer time intervals in macrophages treated with either ␣ 2M* or Pseudomonas exotoxin A. By contrast, when receptor associated protein (RAP) bound to LRP, ubiquitinylation did not occur. While RAP is not found in the extracellular environment it binds to LRP and is believed to function as an intracellular chaperone. The presence of RAP within the cell may, therefore, contribute to the recycling of intact LRP which has ligated and internalized its ligands. © 2001 Elsevier Science Key Words: low density lipoprotein receptor-related protein; receptor ubiquitinylation; macrophage receptor regulation; receptor related protein and receptor ubiquitinylation; ␣ 2-macroglobulin; pseudomonas exotoxin A; protein phosphorylation.
The low density lipoprotein receptor-related protein (LRP) 2 is part of the LDL receptor family, which functions like a scavenger receptor (1). It is synthesized as a ⬃600-kDa single polypeptide chain which is cleaved 1
To whom correspondence and reprint requests should be addressed. Fax: (919) 684-8689. E-mail: [email protected]. 2 Abbreviations used: LRP, low density lipoprotein receptor-related protein; RAP, receptor associated protein; ␣ 2M*, activated forms of ␣ 2-macroglobulin which bind to LRP. 106
to yield two subunits of ⬃515 and ⬃85 kDa in the mature receptor (1). We previously demonstrated that binding of a number of ligands to LRP triggers inositol 1,4,5-trisphosphate-dependent increases in cytosolic free Ca 2⫹ in a process mediated by a pertussis toxin sensitive G protein (2 and references therein). Subsequent studies suggest that the 85-kDa subunit is a transmembrane receptor which associates with a variety of macromolecules involved in transducing signals (2). LRP contains a number of independent binding domains and most of its ligands fail to cross-complete receptor binding (1). The receptor associated protein (RAP) is a significant exception and it blocks the binding of all known LRP ligands (1). Moreover, it also does not induce LRP-mediated signal transduction when it binds to the receptor (3). The function of RAP remains unclear since it does not occur in the extracellular millieu despite the fact that purified receptor always carries some bound RAP (4). When RAP is added to cells, the LRP–RAP complex, is taken up rapidly, and RAP degraded while the receptor recycles, properties shared with other LRP ligands (5, 6). RAP is primarily localized within early compartments of the secretory pathway, including the endoplasmic reticulum (70%), cis-Golgi (24%), and endosomes (4%), while about 2% of the pool is at the cell surface (7). It has been suggested that RAP functions as a molecular chaperone by binding to newly synthesized LRP thereby preventing the binding of potential ligands in various intracellular compartments (8). It has also been reported to play a role in the maturation, folding, and trafficking of LRP to the cell surface (8, 9). LRP, like many other receptors, clusters in coated pits and is internalized constitutively independent of ligand occupancy (10 –12). Many membrane proteins, including LRP, demonstrate long half-lives and recycle back to the plasma membrane; however, others such as 0003-9861/01 $35.00 © 2001 Elsevier Science All rights reserved.
UBIQUITINYLATION OF LRP
various growth factor receptors, are degraded in cytosolic compartments (11, 13, 14). The ubiquitin system is an important mechanism for targeting such proteins for destruction (13–16). This includes proteins of the cytosol, endoplasmic reticulum, plasma membrane, and nucleus (13–16). The process of “marking” proteins with ubiquitin is complex and involves a number of enzyme-catalyzed steps (13–15). Once a protein is ubiquitinylated, it is usually targeted for degradation by the 26S proteosome complex (13–15). Such mechanisms participate in downregulating the growth hormone receptor, the IgE receptor, the platelet-derived growth factor receptor, and the epidermal growth factor receptor to name a few (14 –16). In this study, we report that ligation of LRP by ␣ 2M* or Pseudomonas exotoxin A is followed by formation of ubiquitin-LRP heavy chain, which is targeted for proteosome-mediated degradation. The presence of RAP blocks ubiquitinylation suggesting a previously unknown function for RAP. We suggest that the presence of RAP protects LRP from intracellular destruction and accounts for the fact that LRP can rapidly recycle to the cell surface. MATERIALS AND METHODS Materials. The sources of thioglycollate, cell culture, materials, genestein, chelerythrin, ␣2-macroglobulin-methylamine (␣2M*), Pseudomonas exotoxin A, and RAP have previously been described (2). MG-132 (Z-Leu-Leu-Leu-CHO) and H-89 were purchased from Biomol (Plymouth Meeting, PA). Monoclonal antibody against the light and heavy chains of LRP were purchased from American Diagnostica Inc. (Greenwich, CT). Mouse monoclonal antibody against ubiquitinylated proteins was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). ECF kits were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). All other reagents used were purchased locally and were of the best available grade. Cell lysis, immunoprecipitation, and Western blotting of ubiquitinylated LRP. Thioglycollate-elicited peritoneal macrophages were routinely obtained from pathogen-free 6-week-old C57BI/6 mice (NCI, Frederick, MD) in Hanks’ balanced salt solution containing 10 mM Hepes, pH 7.2, and 3.5 mM NaHCO3 (HHBSS). The cells were washed once with HHBSS, suspended in RPMI 1640 medium containing 2 mM glutamine, 12.5 units/ml penicillin, 6.25 g/ml streptomycin, and 5% bovine serine albumin, plated at a cell density of 3.5– 4 ⫻ 10 6 cells either in 6- or 12-well plates, and incubated for 2 h at 37°C in a humidified CO 2 (5%) incubator. The monolayers were washed twice with HHBSS to remove nonadherent cells, a volume of the above RPMI medium where serum was substituted by 0.2% fatty acid free BSA added and cells incubated overnight as above. Overnight incubated cells were washed with HHBSS, and a volume of RPMI medium added. The cells were treated with the desired concentration of ␣ 2M*, P. exotoxin A, or RAP in separate experiments and incubated at 30°C for various periods of time. The reaction was terminated by aspirating the medium. The cells were washed twice with low pH buffer (50 mM glycine, 150 mM NaCl, 0.1% BSA, pH 2.5) and a volume of lysis buffer containing 20 mM Tris–HCl (pH 8.0), 0.1 M NaCl, 1 mM EDTA, 50 mM NaF, 30 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, 20 g/ml leupeptin, and 0.5% NP-40, was added to the monolayers. The cells were left on ice for 15 min and lysate drawn through a 27-gauge needle three or four times to break DNA strands. Protein in cell lysates was quantified by
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the method of Bradford (17). Equal amounts of lysate protein were employed in a immunoprecipitation either with anti-ubiquitin antibody (1:800 dilution) or for LRP with anti-LRP antibodies (1:800 dilution). Immunoprecipitation, PAGE, Western blotting, and the detection and quantitation of protein on the membrane with ECF kit was done according to the suppliers instructions, using a phosphorimager (Storm 800). Modulation of ␣ 2M*-induced ubiquitinylation of LRP. In experiments where the effect of the proteosome inhibitor MG-132 (20 M/18 h) (18), the protein tyrosine kinase inhibitor genestein (20 M/16 h) (19), the protein kinase C inhibitor, chelerythrin (200 nM/15 min) (20), or the c-AMP-dependent protein kinase inhibitor H-89 (15 M/3 h) (21) on ␣ 2M*-induced ubiquitinylation were examined the cells were preincubated with the respective inhibitors before stimulating with ␣ 2M*. The monolayers were washed and stimulated with respective ligands, incubated as above, and processed for Western blotting and detection of ubiquitinylated proteins as described above.
RESULTS
Ligation of LRP with ␣ 2M* or pseudomonas exotoxin A induces ubiquitinylation of LRP. Exposure of macrophages to two ligands of LRP, ␣ 2M* or Pseudomonas exotoxin A promoted ubiquitinylation of LRP heavy chain (Fig. 1A). Ubiquitinylation of LRP is dose-dependent as shown by studies with ␣ 2M* (Figs. 1B and 1C). The maximum detection of ubiquitin-LRP occurred at a ligand concentration of ⬃0.5 to 1.0 M ␣ 2M*. Thereafter steady state was achieved. Ubiquitinylation of LRP was determined by immunoblotting of the macrophage cell lysates after ligand exposure. In all cases, the blot was reprobed for LRP with specific monoclonal antibodies to confirm the identification of the LRP heavy and light chains. A time course study was also performed with ␣ 2M*. As can be seen (Fig. 2) the maximal level of ubiquitinLRP heavy was present at 25 to 30 min of incubation of the macrophages with ␣ 2M* at 30°C. Levels of detectable LRP and ␣ 2M* paralleled levels of ubiquitinylation of LRP, suggesting that the ubiquitinylated LRP/ ␣ 2M* complex was subject to degradation, consistent with the typical behavior of other proteins that are so modified (13–16). Ligation of LRP with RAP does not induce ubiquitinylation of LRP. Macrophages were then treated with various concentrations of RAP for 30 min at 30°C (Fig. 3A). By contrast to studies described above, LRP from these cell lysates showed very little ubiquitinylation of the receptor. The most obvious interpretation of these results is that uptake of LRP with RAP bound to the receptor blocks ubiquitinylation. It is, however, also possible that RAP favors more rapid proteosome-mediated degradation. In order to address these issues, MG-132 was employed to inhibit proteosome-complex mediated degradation of ubiquitin-LRP. Macrophages were pretreated with this inhibitor (20 M/16 h) prior to exposure to either ␣ 2M* or RAP (Fig. 3B). As can be seen, this pretreatment prolonged the detectability of
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ubiquitin-LRP in ␣ 2M*-treated cells. No such effect was observed in the RAP exposed macrophages. It is concluded that LRP bound to RAP is protected from ubiquitinylation and, therefore, subsequent proteosome-mediated degradation. Inhibition of kinase activity suppresses ubiquitinylation of LRP. Previous studies have suggested that kinase activation may be associated with ubquitinylation of target proteins (13–16). In the next studies, therefore, we examined the role of Tyr and Ser/Thr phosphorylation in ligand-induced ubiquitinylation of LRP (Fig. 4). Specifically, prior to exposure, ␣ 2M* macrophages were pretreated with genestein, a Tyr kinase
FIG. 1. Immunoblots of ubiquitinylated and nonubiquitinylated LRP heavy chain (⬃515 kDa) and light chain (⬃85 kDa) in ␣ 2 M*stimulated macrophages. (A) Lane 1, Immunoblot of LRP heavy chain (⬃515 kDa); lane 2, immunoblot of LRP light chain (⬃85 kDa); lanes 3–5 immunoblots of ubiquitinylated LRP in buffer (lane 3), ␣ 2 M* (1 nM/25 min) lane (4), and P. Exotoxin A (10 g/ml/25 min) (lane 5) exposed macrophages. Immunoblots of ubiquitinylated LRP were reprobed with antibody against the heavy chain and light chain of LRP demonstrating that only the heavy chain is modified. The data are representative of four or five individual experiments. (B) Effect of ␣ 2 M* concentration on production of ubiquitinylated LRP heavy chain. The data shown are representative of four individual experiments. The concentration of ␣ 2 M* was: lane 1, 0; lanes 2– 8, 25, 50, 100, 200, 500, 1000, or 2000 pM, respectively. (C) ␣ 2 M* concentration-dependent formation of ubiquitinylated LRP heavy chain at 25 min of incubation. The graph is derived from B by quantifying the immunoblots by Phosphorimager. Values are mean ⫾ SE from four independent experiments and are expressed as the percentage of LRP that is ubiquitinylated in ␣ 2 M*-exposed cells.
FIG. 2. Effect of time of incubation of macrophages with ␣ 2M* on ubiquitinylated LRP heavy chain. (A) Ubiquitinylated LRP heavy chain. (B) LRP heavy chain and ␣ 2M* detected after stripping and reprobing the membrane from A with antibodies against the heavy chain of LRP and ␣ 2M*. The data shown are representative of three individual experiments. (C) Graphical representation of the duration of persistence of ubiquitinylated LRP heavy chain, the modified LRP heavy chain, and ␣ 2M*. The immunoblots from A and B were quantified individually by phosphorimager. The results are expressed as percentage of ubiquitinylated LRP heavy chain, LRP heavy chain, and ␣ 2M* formed in ␣ 2M*-exposed cells. The percentage of ubiquitinylated LRP was calculated as described in the legend of Fig. 1.
UBIQUITINYLATION OF LRP
FIG. 3. Effect of stimulation of macrophages with varying concentration of RAP on ubiquitinylation of the heavy chain of LRP. (A) Immunoblot of ubiquitinylated LRP, which colocalized with immunoblots obtained with LRP heavy chain antibody on reprobing the membranes. The data shown are representative of three independent experiments. (B) Effect of proteosome inhibitor MG-132 on the generation of ubiquitinylated LRP heavy chain in macrophages treated with ␣ 2M* or RAP: (F) Ubiquinylated LRP heavy chain in ␣ 2M* exposed cells; (E) ubiquitinylated LRP heavy chain in cells pretreated with MG-132 before ␣ 2M* stimulation; (Œ) ubiquitinylated LRP heavy chain in RAP treated cells; (‚) ubiquitinylated LRP heavy chain in cells pretreated with MG-132 before RAP treatment. Values are expressed as the percentage of LRP that is ubiquitinylated and are the mean ⫾ SE from four individual experiments.
inhibitor, chelerythrin, a protein kinase C inhibitor, or H-89, a cAMP-dependent protein kinase A inhibitor. Inhibition of each of these kinases affected the ubiquitinylation of LRP after ␣ 2M* exposure. The greatest effects were seen after cells were exposured to chlerythrin or H-89. DISCUSSION
In this study, we demonstrate that ligation of LRP by ␣ 2M* or Pseudomonas exotoxin A promotes receptor ubiquitinylation and subsequent proteosome-mediated degradation. However, when LRP is ligated by RAP, the receptor demonstrates minimal ubiquitinylation and proteosome-mediated degradation. The role of
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RAP in regulating LRP function is a question of considerable interest. Previous studies have focused on the role of RAP as a molecular chaperone allowing LRP to reach the surface of cells properly folded and not having bound an intracellular ligand (7, 8). The latter is an important issue since LRP has such a broad range of potential ligands (1). LRP is recognized as a receptor which recycles after uptake via clathrin coated pits (10 –12). Bound ligands are targeted to the lysozomes for degradation (10 –12). The present study is the first to our knowledge which suggests that at least a fraction of the LRP so internalized is ubiquitinylated and targeted for proteosomeal degradation. Of interest, is the observation that only the LRP heavy chain, M r ⬃515,000, is ubiquitinylated. This subunit contains all of the ligand binding domains of the receptor (1). The light chain, M r ⬃85,000, is anchored to the membrane. A recent study has suggested that differential trafficking of these two subunits occurs after ligation of the receptor (22). It is likely that the specificity of ubiquitinylation observed in the present study contributes to this differential handling of the two subunits. We suggest that the availability of RAP in various intracellular compartments can replace other LRPbound ligands and protect the receptor from ubiquitinylation and degradation. This allows intact LRP to recycle to the cell surface. Whether LRP, which binds to other members of the LDL receptor family, regulates recycling of these receptors is an interesting question for future studies.
FIG. 4. Effect of protein phosphorylation on ␣ 2M* induced ubiquitinylation of LRP heavy chain. Experimental details are given under Materials and Methods. The bars are: (1) buffer-treated controls; (2) ␣ 2M* (1 nM/25 min)-treated; (3) genestein (20 M/16 h) then ␣ 2M* (1 nM/25 min); (4) H-89 (2 M/3 h) then ␣ 2M* (1 nM/25 min); and (5) chelerythrin (200 nM/15 min) then ␣ 2M* (1 nM/25 min). LRP heavy chain was detected by reprobing the immunoblots with antibody against the heavy chain of LRP. The immunoblots were quantified by phosphorimager and the results are expressed as the percentage of LRP that is ubiquitinylated and are the mean ⫾ SE from three independent experiments.
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In general, three enzyme-mediated steps are required to ubiquitinylate a target protein (13–15). After activation of ubiquitin forming an E1-SOCAO-ubiquitin moiety, the ubiquitin is transferred to the active site Cys residue of a carrier protein, E2. A ubiquitinprotein ligase (E3) then catallyzes transfer of ubiquitin to the target protein forming a peptide bond between the COOH terminal of ubiquitin and -amino group of lysine residues of the target protein (13–15). Based on our data, we propose that RAP binding to LRP blocks the third step in this reaction sequence; namely, transfer of ubiquitin to the receptor. In this regard it is interesting to note that RAP blocks the binding to the receptor of all known LRP ligands (1). This behavior is unusual, since very few ligands are able to cross-compete for binding to LRP which consists of a number of independent binding domains (1). These observations have led to the logical hypothesis that RAP blocks ligand interaction with all LRP domains by inducing a conformational change in the receptor thus promoting dissociation of bound ligands and blocking access to as yet unbound ligands (4, 23, 24). Such a conformational change may also block access of critical LRP lysine residues to the action of the ubiquitin lyase. In the present study, we have also shown that activation of kinases is important in promoting ubiquitinylation of LRP. It is unclear whether the phosphorylation reactions involved affect the target LRP or are necessary for activating the ubiquitinylation enzymes (13–16). In this regard it is known that LRP is phosphorylated during its internalization (25, 26 and U. K. Misra and S. V. Pizzo, unpublished data). It is, therefore, conceivable that the phosphorylation of LRP is a prerequisitive for its ubiquitinylation. ACKNOWLEDGMENT This work was supported by National Heart, Lung, and Blood Institute Grant HL-24066.
REFERENCES 1. Krieger, M., and Herz, J. (1994) Annu. Rev. Biochem. 63, 601– 638. 2. Misra, U. K., Gawdi, G., and Pizzo, S. V. (1999) Arch. Biochem. Biophys. 372(2), 238 –247.
3. Misra, U. K., Chu, C., Gawdi, G., and Pizzo, S. V. (1994) J. Biol. Chem. 269, 12541–12547. 4. Striekland, D. K., Ashcom, J. D., Williams, S., Battey, F., Behre, E., McTigue, K., Battey, J. F., and Argraves, W. S. (1991) J. Biol. Chem. 266(20), 13364 –13369. 5. Herz, J., Goldstein, J. L., Strickland, D. K., Ho, Y. K., and Brown, M. S. (1991) J. Biol. Chem. 266(31), 21232–21238. 6. Iadonato, S. P., Bu, G., Maksymovitch, E. A., and Schwartz, A. L. (1993) Biochem. J. 296(3), 867– 875. 7. Bu, G., Maksymovitch, E. A., Geuze, H., and Schwartz, A. L. (1994) J. Biol. Chem. 269(47), 29874 –29882. 8. Bu, G., and Rennke, S. (1996) J. Biol. Chem. 271(36), 22218 – 22224. 9. Bu, G., and Schwartz, A. L. (1998) Trends Cell Biol. 8(7), 272– 276. 10. Kaplan, J., and Nielsen, M. L. (1979) J. Biol. Chem. 254(15), 7329 –7335. 11. Mellman, I. (1996) Annu. Rev. Cell Dev. Biol. 12, 575– 625. 12. Hussain, M. M., Strickland, D. K., and Bakillah, A. (1999) Annu. Rev. Nutr. 19, 141–172. 13. Hicke, L. (1999) Trends Cell Biol. 9(3), 107–112. 14. Hershko, A., and Ciechanover, A. (1998) Annu. Rev. Biochem. 67, 425– 479. 15. Hicke, L. (1997) Faseb J. 11(14), 1215–1226. 16. van Kerkhof, P., Sachse, M., Klumperman, J., and Strous, G. J. (2001) J. Biol. Chem. 276(6), 3778 –3784. 17. Bradford, M. M. (1976) Anal. Biochem. 72, 248 –254. 18. Alves dos Santos, C. M., van Kerkhof, P., and Strous, G. J. (2001) J. Biol. Chem. 276(14), 10839 –10846. 19. Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M., and Fukami, Y. (1987) J. Biol. Chem. 262(12), 5592–5595. 20. Herbert, J. M., Augereau, J. M., Gleye, J., and Maffrand, J. P. (1990) Biochem. Biophys. Res. Commun. 172(3), 993–999. 21. Chijiwa, T., Mishima, A., Hagiwara, M., Sano, M., Hayashi, K., Inoue, T., Naito, K., Toshioka, T., and Hidaka, H. (1990) J. Biol. Chem. 265(9), 5267–5272. 22. Herz, J., Kowal, R. C., Ho, Y. K., Brown, M. S., and Goldstein, J. L. (1990) J. Biol. Chem. 265(34), 21355–21362. 23. Warshawsky, I., Bu, G., and Schwartz, A. L. (1993) J. Biol. Chem. 268(29), 22046 –22054. 24. Medved, L. V., Migliorini, M., Mikhailenko, I., Barrientos, L. G., Llinas, M., and Strickland, D. K. (1999) J. Biol. Chem. 274(2), 717–727. 25. Chen, W. J., Goldstein, J. L., and Brown, M. S. (1990) J. Biol. Chem. 265(6), 3116 –3123. 26. Barnes, H., Larsen, B., Tyers, M., and van Der Geer, P. (2001) J. Biol. Chem. 276(22), 19119 –19125.
Receptor-Associated Protein Binding Blocks Ubiquitinylation of the Low Density Lipoprotein Receptor-Related Protein U. K. Misra and S. V. Pizzo 1 Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710
Received August 16, 2001
The low density lipoprotein receptor-related protein (LRP) consists of two subunits, M r ⬃ 515,000 and 85,000. LRP is a receptor for activated ␣ 2-macrogobulin (␣ 2M*), Pseudomonas exotoxin A, and many other proteins. We now report that ubiquitinylation of the LRP heavy chain occurred when either Pseudomonas exotoxin A or ␣ 2M* bound to LRP on macrophages. Ubiquitinylation was dose-dependent and maximal about 30 min after ligation of the receptor. Addition of the proteosome inhibitor MG-132 sustained the level of ubiquitin-LRP for longer time intervals in macrophages treated with either ␣ 2M* or Pseudomonas exotoxin A. By contrast, when receptor associated protein (RAP) bound to LRP, ubiquitinylation did not occur. While RAP is not found in the extracellular environment it binds to LRP and is believed to function as an intracellular chaperone. The presence of RAP within the cell may, therefore, contribute to the recycling of intact LRP which has ligated and internalized its ligands. © 2001 Elsevier Science Key Words: low density lipoprotein receptor-related protein; receptor ubiquitinylation; macrophage receptor regulation; receptor related protein and receptor ubiquitinylation; ␣ 2-macroglobulin; pseudomonas exotoxin A; protein phosphorylation.
The low density lipoprotein receptor-related protein (LRP) 2 is part of the LDL receptor family, which functions like a scavenger receptor (1). It is synthesized as a ⬃600-kDa single polypeptide chain which is cleaved 1
To whom correspondence and reprint requests should be addressed. Fax: (919) 684-8689. E-mail: [email protected]. 2 Abbreviations used: LRP, low density lipoprotein receptor-related protein; RAP, receptor associated protein; ␣ 2M*, activated forms of ␣ 2-macroglobulin which bind to LRP. 106
to yield two subunits of ⬃515 and ⬃85 kDa in the mature receptor (1). We previously demonstrated that binding of a number of ligands to LRP triggers inositol 1,4,5-trisphosphate-dependent increases in cytosolic free Ca 2⫹ in a process mediated by a pertussis toxin sensitive G protein (2 and references therein). Subsequent studies suggest that the 85-kDa subunit is a transmembrane receptor which associates with a variety of macromolecules involved in transducing signals (2). LRP contains a number of independent binding domains and most of its ligands fail to cross-complete receptor binding (1). The receptor associated protein (RAP) is a significant exception and it blocks the binding of all known LRP ligands (1). Moreover, it also does not induce LRP-mediated signal transduction when it binds to the receptor (3). The function of RAP remains unclear since it does not occur in the extracellular millieu despite the fact that purified receptor always carries some bound RAP (4). When RAP is added to cells, the LRP–RAP complex, is taken up rapidly, and RAP degraded while the receptor recycles, properties shared with other LRP ligands (5, 6). RAP is primarily localized within early compartments of the secretory pathway, including the endoplasmic reticulum (70%), cis-Golgi (24%), and endosomes (4%), while about 2% of the pool is at the cell surface (7). It has been suggested that RAP functions as a molecular chaperone by binding to newly synthesized LRP thereby preventing the binding of potential ligands in various intracellular compartments (8). It has also been reported to play a role in the maturation, folding, and trafficking of LRP to the cell surface (8, 9). LRP, like many other receptors, clusters in coated pits and is internalized constitutively independent of ligand occupancy (10 –12). Many membrane proteins, including LRP, demonstrate long half-lives and recycle back to the plasma membrane; however, others such as 0003-9861/01 $35.00 © 2001 Elsevier Science All rights reserved.
UBIQUITINYLATION OF LRP
various growth factor receptors, are degraded in cytosolic compartments (11, 13, 14). The ubiquitin system is an important mechanism for targeting such proteins for destruction (13–16). This includes proteins of the cytosol, endoplasmic reticulum, plasma membrane, and nucleus (13–16). The process of “marking” proteins with ubiquitin is complex and involves a number of enzyme-catalyzed steps (13–15). Once a protein is ubiquitinylated, it is usually targeted for degradation by the 26S proteosome complex (13–15). Such mechanisms participate in downregulating the growth hormone receptor, the IgE receptor, the platelet-derived growth factor receptor, and the epidermal growth factor receptor to name a few (14 –16). In this study, we report that ligation of LRP by ␣ 2M* or Pseudomonas exotoxin A is followed by formation of ubiquitin-LRP heavy chain, which is targeted for proteosome-mediated degradation. The presence of RAP blocks ubiquitinylation suggesting a previously unknown function for RAP. We suggest that the presence of RAP protects LRP from intracellular destruction and accounts for the fact that LRP can rapidly recycle to the cell surface. MATERIALS AND METHODS Materials. The sources of thioglycollate, cell culture, materials, genestein, chelerythrin, ␣2-macroglobulin-methylamine (␣2M*), Pseudomonas exotoxin A, and RAP have previously been described (2). MG-132 (Z-Leu-Leu-Leu-CHO) and H-89 were purchased from Biomol (Plymouth Meeting, PA). Monoclonal antibody against the light and heavy chains of LRP were purchased from American Diagnostica Inc. (Greenwich, CT). Mouse monoclonal antibody against ubiquitinylated proteins was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). ECF kits were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). All other reagents used were purchased locally and were of the best available grade. Cell lysis, immunoprecipitation, and Western blotting of ubiquitinylated LRP. Thioglycollate-elicited peritoneal macrophages were routinely obtained from pathogen-free 6-week-old C57BI/6 mice (NCI, Frederick, MD) in Hanks’ balanced salt solution containing 10 mM Hepes, pH 7.2, and 3.5 mM NaHCO3 (HHBSS). The cells were washed once with HHBSS, suspended in RPMI 1640 medium containing 2 mM glutamine, 12.5 units/ml penicillin, 6.25 g/ml streptomycin, and 5% bovine serine albumin, plated at a cell density of 3.5– 4 ⫻ 10 6 cells either in 6- or 12-well plates, and incubated for 2 h at 37°C in a humidified CO 2 (5%) incubator. The monolayers were washed twice with HHBSS to remove nonadherent cells, a volume of the above RPMI medium where serum was substituted by 0.2% fatty acid free BSA added and cells incubated overnight as above. Overnight incubated cells were washed with HHBSS, and a volume of RPMI medium added. The cells were treated with the desired concentration of ␣ 2M*, P. exotoxin A, or RAP in separate experiments and incubated at 30°C for various periods of time. The reaction was terminated by aspirating the medium. The cells were washed twice with low pH buffer (50 mM glycine, 150 mM NaCl, 0.1% BSA, pH 2.5) and a volume of lysis buffer containing 20 mM Tris–HCl (pH 8.0), 0.1 M NaCl, 1 mM EDTA, 50 mM NaF, 30 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, 20 g/ml leupeptin, and 0.5% NP-40, was added to the monolayers. The cells were left on ice for 15 min and lysate drawn through a 27-gauge needle three or four times to break DNA strands. Protein in cell lysates was quantified by
107
the method of Bradford (17). Equal amounts of lysate protein were employed in a immunoprecipitation either with anti-ubiquitin antibody (1:800 dilution) or for LRP with anti-LRP antibodies (1:800 dilution). Immunoprecipitation, PAGE, Western blotting, and the detection and quantitation of protein on the membrane with ECF kit was done according to the suppliers instructions, using a phosphorimager (Storm 800). Modulation of ␣ 2M*-induced ubiquitinylation of LRP. In experiments where the effect of the proteosome inhibitor MG-132 (20 M/18 h) (18), the protein tyrosine kinase inhibitor genestein (20 M/16 h) (19), the protein kinase C inhibitor, chelerythrin (200 nM/15 min) (20), or the c-AMP-dependent protein kinase inhibitor H-89 (15 M/3 h) (21) on ␣ 2M*-induced ubiquitinylation were examined the cells were preincubated with the respective inhibitors before stimulating with ␣ 2M*. The monolayers were washed and stimulated with respective ligands, incubated as above, and processed for Western blotting and detection of ubiquitinylated proteins as described above.
RESULTS
Ligation of LRP with ␣ 2M* or pseudomonas exotoxin A induces ubiquitinylation of LRP. Exposure of macrophages to two ligands of LRP, ␣ 2M* or Pseudomonas exotoxin A promoted ubiquitinylation of LRP heavy chain (Fig. 1A). Ubiquitinylation of LRP is dose-dependent as shown by studies with ␣ 2M* (Figs. 1B and 1C). The maximum detection of ubiquitin-LRP occurred at a ligand concentration of ⬃0.5 to 1.0 M ␣ 2M*. Thereafter steady state was achieved. Ubiquitinylation of LRP was determined by immunoblotting of the macrophage cell lysates after ligand exposure. In all cases, the blot was reprobed for LRP with specific monoclonal antibodies to confirm the identification of the LRP heavy and light chains. A time course study was also performed with ␣ 2M*. As can be seen (Fig. 2) the maximal level of ubiquitinLRP heavy was present at 25 to 30 min of incubation of the macrophages with ␣ 2M* at 30°C. Levels of detectable LRP and ␣ 2M* paralleled levels of ubiquitinylation of LRP, suggesting that the ubiquitinylated LRP/ ␣ 2M* complex was subject to degradation, consistent with the typical behavior of other proteins that are so modified (13–16). Ligation of LRP with RAP does not induce ubiquitinylation of LRP. Macrophages were then treated with various concentrations of RAP for 30 min at 30°C (Fig. 3A). By contrast to studies described above, LRP from these cell lysates showed very little ubiquitinylation of the receptor. The most obvious interpretation of these results is that uptake of LRP with RAP bound to the receptor blocks ubiquitinylation. It is, however, also possible that RAP favors more rapid proteosome-mediated degradation. In order to address these issues, MG-132 was employed to inhibit proteosome-complex mediated degradation of ubiquitin-LRP. Macrophages were pretreated with this inhibitor (20 M/16 h) prior to exposure to either ␣ 2M* or RAP (Fig. 3B). As can be seen, this pretreatment prolonged the detectability of
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ubiquitin-LRP in ␣ 2M*-treated cells. No such effect was observed in the RAP exposed macrophages. It is concluded that LRP bound to RAP is protected from ubiquitinylation and, therefore, subsequent proteosome-mediated degradation. Inhibition of kinase activity suppresses ubiquitinylation of LRP. Previous studies have suggested that kinase activation may be associated with ubquitinylation of target proteins (13–16). In the next studies, therefore, we examined the role of Tyr and Ser/Thr phosphorylation in ligand-induced ubiquitinylation of LRP (Fig. 4). Specifically, prior to exposure, ␣ 2M* macrophages were pretreated with genestein, a Tyr kinase
FIG. 1. Immunoblots of ubiquitinylated and nonubiquitinylated LRP heavy chain (⬃515 kDa) and light chain (⬃85 kDa) in ␣ 2 M*stimulated macrophages. (A) Lane 1, Immunoblot of LRP heavy chain (⬃515 kDa); lane 2, immunoblot of LRP light chain (⬃85 kDa); lanes 3–5 immunoblots of ubiquitinylated LRP in buffer (lane 3), ␣ 2 M* (1 nM/25 min) lane (4), and P. Exotoxin A (10 g/ml/25 min) (lane 5) exposed macrophages. Immunoblots of ubiquitinylated LRP were reprobed with antibody against the heavy chain and light chain of LRP demonstrating that only the heavy chain is modified. The data are representative of four or five individual experiments. (B) Effect of ␣ 2 M* concentration on production of ubiquitinylated LRP heavy chain. The data shown are representative of four individual experiments. The concentration of ␣ 2 M* was: lane 1, 0; lanes 2– 8, 25, 50, 100, 200, 500, 1000, or 2000 pM, respectively. (C) ␣ 2 M* concentration-dependent formation of ubiquitinylated LRP heavy chain at 25 min of incubation. The graph is derived from B by quantifying the immunoblots by Phosphorimager. Values are mean ⫾ SE from four independent experiments and are expressed as the percentage of LRP that is ubiquitinylated in ␣ 2 M*-exposed cells.
FIG. 2. Effect of time of incubation of macrophages with ␣ 2M* on ubiquitinylated LRP heavy chain. (A) Ubiquitinylated LRP heavy chain. (B) LRP heavy chain and ␣ 2M* detected after stripping and reprobing the membrane from A with antibodies against the heavy chain of LRP and ␣ 2M*. The data shown are representative of three individual experiments. (C) Graphical representation of the duration of persistence of ubiquitinylated LRP heavy chain, the modified LRP heavy chain, and ␣ 2M*. The immunoblots from A and B were quantified individually by phosphorimager. The results are expressed as percentage of ubiquitinylated LRP heavy chain, LRP heavy chain, and ␣ 2M* formed in ␣ 2M*-exposed cells. The percentage of ubiquitinylated LRP was calculated as described in the legend of Fig. 1.
UBIQUITINYLATION OF LRP
FIG. 3. Effect of stimulation of macrophages with varying concentration of RAP on ubiquitinylation of the heavy chain of LRP. (A) Immunoblot of ubiquitinylated LRP, which colocalized with immunoblots obtained with LRP heavy chain antibody on reprobing the membranes. The data shown are representative of three independent experiments. (B) Effect of proteosome inhibitor MG-132 on the generation of ubiquitinylated LRP heavy chain in macrophages treated with ␣ 2M* or RAP: (F) Ubiquinylated LRP heavy chain in ␣ 2M* exposed cells; (E) ubiquitinylated LRP heavy chain in cells pretreated with MG-132 before ␣ 2M* stimulation; (Œ) ubiquitinylated LRP heavy chain in RAP treated cells; (‚) ubiquitinylated LRP heavy chain in cells pretreated with MG-132 before RAP treatment. Values are expressed as the percentage of LRP that is ubiquitinylated and are the mean ⫾ SE from four individual experiments.
inhibitor, chelerythrin, a protein kinase C inhibitor, or H-89, a cAMP-dependent protein kinase A inhibitor. Inhibition of each of these kinases affected the ubiquitinylation of LRP after ␣ 2M* exposure. The greatest effects were seen after cells were exposured to chlerythrin or H-89. DISCUSSION
In this study, we demonstrate that ligation of LRP by ␣ 2M* or Pseudomonas exotoxin A promotes receptor ubiquitinylation and subsequent proteosome-mediated degradation. However, when LRP is ligated by RAP, the receptor demonstrates minimal ubiquitinylation and proteosome-mediated degradation. The role of
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RAP in regulating LRP function is a question of considerable interest. Previous studies have focused on the role of RAP as a molecular chaperone allowing LRP to reach the surface of cells properly folded and not having bound an intracellular ligand (7, 8). The latter is an important issue since LRP has such a broad range of potential ligands (1). LRP is recognized as a receptor which recycles after uptake via clathrin coated pits (10 –12). Bound ligands are targeted to the lysozomes for degradation (10 –12). The present study is the first to our knowledge which suggests that at least a fraction of the LRP so internalized is ubiquitinylated and targeted for proteosomeal degradation. Of interest, is the observation that only the LRP heavy chain, M r ⬃515,000, is ubiquitinylated. This subunit contains all of the ligand binding domains of the receptor (1). The light chain, M r ⬃85,000, is anchored to the membrane. A recent study has suggested that differential trafficking of these two subunits occurs after ligation of the receptor (22). It is likely that the specificity of ubiquitinylation observed in the present study contributes to this differential handling of the two subunits. We suggest that the availability of RAP in various intracellular compartments can replace other LRPbound ligands and protect the receptor from ubiquitinylation and degradation. This allows intact LRP to recycle to the cell surface. Whether LRP, which binds to other members of the LDL receptor family, regulates recycling of these receptors is an interesting question for future studies.
FIG. 4. Effect of protein phosphorylation on ␣ 2M* induced ubiquitinylation of LRP heavy chain. Experimental details are given under Materials and Methods. The bars are: (1) buffer-treated controls; (2) ␣ 2M* (1 nM/25 min)-treated; (3) genestein (20 M/16 h) then ␣ 2M* (1 nM/25 min); (4) H-89 (2 M/3 h) then ␣ 2M* (1 nM/25 min); and (5) chelerythrin (200 nM/15 min) then ␣ 2M* (1 nM/25 min). LRP heavy chain was detected by reprobing the immunoblots with antibody against the heavy chain of LRP. The immunoblots were quantified by phosphorimager and the results are expressed as the percentage of LRP that is ubiquitinylated and are the mean ⫾ SE from three independent experiments.
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In general, three enzyme-mediated steps are required to ubiquitinylate a target protein (13–15). After activation of ubiquitin forming an E1-SOCAO-ubiquitin moiety, the ubiquitin is transferred to the active site Cys residue of a carrier protein, E2. A ubiquitinprotein ligase (E3) then catallyzes transfer of ubiquitin to the target protein forming a peptide bond between the COOH terminal of ubiquitin and -amino group of lysine residues of the target protein (13–15). Based on our data, we propose that RAP binding to LRP blocks the third step in this reaction sequence; namely, transfer of ubiquitin to the receptor. In this regard it is interesting to note that RAP blocks the binding to the receptor of all known LRP ligands (1). This behavior is unusual, since very few ligands are able to cross-compete for binding to LRP which consists of a number of independent binding domains (1). These observations have led to the logical hypothesis that RAP blocks ligand interaction with all LRP domains by inducing a conformational change in the receptor thus promoting dissociation of bound ligands and blocking access to as yet unbound ligands (4, 23, 24). Such a conformational change may also block access of critical LRP lysine residues to the action of the ubiquitin lyase. In the present study, we have also shown that activation of kinases is important in promoting ubiquitinylation of LRP. It is unclear whether the phosphorylation reactions involved affect the target LRP or are necessary for activating the ubiquitinylation enzymes (13–16). In this regard it is known that LRP is phosphorylated during its internalization (25, 26 and U. K. Misra and S. V. Pizzo, unpublished data). It is, therefore, conceivable that the phosphorylation of LRP is a prerequisitive for its ubiquitinylation. ACKNOWLEDGMENT This work was supported by National Heart, Lung, and Blood Institute Grant HL-24066.
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