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Involvement of Heat Shock Protein 90 in the Degradation of Mutant Insulin Receptors by the Proteasome

1998; Elsevier BV; Volume: 273; Issue: 18 Linguagem: Inglês

10.1074/jbc.273.18.11183

1083-351X

Takeshi Imamura, Tetsuro Haruta, Yasumitsu Takata, Isao Usui, Minoru Iwata, Hajime Ishihara, Manabu Ishiki, Osamu Ishibashi, Eiichi Ueno, Toshiyasu Sasaoka, Masashi Kobayashi,

Toxin Mechanisms and Immunotoxins

We previously reported three families with type A insulin-resistant syndrome who had mutations, either Asp1179 or Leu1193, in the kinase domain of the insulin receptor. The extreme insulin resistance of these patients was found to be caused by the decreased number of insulin receptors on the cell surface, due to the intracellular rapid degradation (Imamura, T., Takata, Y., Sasaoka, T., Takada, Y., Morioka, H., Haruta, T., Sawa, T., Iwanishi, M., Yang, G. H., Suzuki, Y., Hamada, J., and Kobayashi, M. (1994) J. Biol. Chem.269, 31019–31027). In the present study, we first examined whether these mutations caused rapid degradation of unprocessed proreceptors, using the exon 13 deleted mutant insulin receptors (ΔEx13-IR), which were accumulated in the endoplasmic reticulum as unprocessed proreceptors. The addition of Asp1179 or Leu1193 mutation to ΔEx13-IR caused accelerated degradation of the unprocessed ΔEx13-IR in the transfected COS-7 cells. Next, we tested whether these mutant receptors were degraded by the proteasome. Treatment with proteasome inhibitors Z-Leu-Leu-Nva-H (MG-115) or Z-Leu-Leu-Leu-H (MG-132) prevented the accelerated degradation of these mutant receptors, resulting in increased amounts of the mutant receptors in the COS-7 cells. Essentially the same results were obtained in the patient's transformed lymphocytes. Finally, we found that these mutant receptors bound to heat shock protein 90 (Hsp90). To determine whether Hsp90 played an important role in the accelerated receptor degradation, we examined the effect of anti-Hsp90 antibody on the mutant receptor degradation. The microinjection of anti-Hsp90 antibody into cells prevented the accelerated degradation of both Asp1179 and Leu1193 mutant insulin receptors. Taken together, these results suggest that Hsp90 is involved in dislocation of the mutant insulin receptors out of the endoplasmic reticulum into the cytosol, where the mutant receptors are degraded by the proteasome. We previously reported three families with type A insulin-resistant syndrome who had mutations, either Asp1179 or Leu1193, in the kinase domain of the insulin receptor. The extreme insulin resistance of these patients was found to be caused by the decreased number of insulin receptors on the cell surface, due to the intracellular rapid degradation (Imamura, T., Takata, Y., Sasaoka, T., Takada, Y., Morioka, H., Haruta, T., Sawa, T., Iwanishi, M., Yang, G. H., Suzuki, Y., Hamada, J., and Kobayashi, M. (1994) J. Biol. Chem.269, 31019–31027). In the present study, we first examined whether these mutations caused rapid degradation of unprocessed proreceptors, using the exon 13 deleted mutant insulin receptors (ΔEx13-IR), which were accumulated in the endoplasmic reticulum as unprocessed proreceptors. The addition of Asp1179 or Leu1193 mutation to ΔEx13-IR caused accelerated degradation of the unprocessed ΔEx13-IR in the transfected COS-7 cells. Next, we tested whether these mutant receptors were degraded by the proteasome. Treatment with proteasome inhibitors Z-Leu-Leu-Nva-H (MG-115) or Z-Leu-Leu-Leu-H (MG-132) prevented the accelerated degradation of these mutant receptors, resulting in increased amounts of the mutant receptors in the COS-7 cells. Essentially the same results were obtained in the patient's transformed lymphocytes. Finally, we found that these mutant receptors bound to heat shock protein 90 (Hsp90). To determine whether Hsp90 played an important role in the accelerated receptor degradation, we examined the effect of anti-Hsp90 antibody on the mutant receptor degradation. The microinjection of anti-Hsp90 antibody into cells prevented the accelerated degradation of both Asp1179 and Leu1193 mutant insulin receptors. Taken together, these results suggest that Hsp90 is involved in dislocation of the mutant insulin receptors out of the endoplasmic reticulum into the cytosol, where the mutant receptors are degraded by the proteasome. Various mutations in the insulin receptor gene have been reported in patients with severe insulin resistance (1Taylor S.I. Diabetes. 1992; 41: 1473-1490Crossref PubMed Scopus (0) Google Scholar). Analysis of the mutated insulin receptors in the cells gave us the opportunity to understand the function and processing of insulin receptor protein. Among the various mechanisms for the insulin resistance in these patients, certain patients with the insulin receptor mutations showed a reduced number of insulin receptors on the cell surface although the mRNA of insulin receptor was normally expressed (1Taylor S.I. Diabetes. 1992; 41: 1473-1490Crossref PubMed Scopus (0) Google Scholar). Two major causes for this phenomenon have been described; the first is the impaired protein processing of mutated insulin receptors and accumulation in the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; Hsp, heat shock protein; Hsc, heat shock cognate; BiP, immunoglobulin heavy chain binding protein; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; IRc, cytoplasmic domain of insulin receptor. 1The abbreviations used are: ER, endoplasmic reticulum; Hsp, heat shock protein; Hsc, heat shock cognate; BiP, immunoglobulin heavy chain binding protein; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; IRc, cytoplasmic domain of insulin receptor.(3Kadowaki T. Kadowaki H. Accili D. Taylor S.I. J. Biol. Chem. 1990; 265: 19143-19150Abstract Full Text PDF PubMed Google Scholar, 4Wertheimer E. Barbetti F. Muggeo M. Roth J. Taylor S.I. J. Biol. Chem. 1994; 269: 7587-7592Abstract Full Text PDF PubMed Google Scholar, 5Van der Vorm E.R. Van der Zon G.C.M. Moller W. Krans H.M.J. Lindhout D. Maassen J.A. J. Biol. Chem. 1992; 267: 66-71Abstract Full Text PDF PubMed Google Scholar, 6Longo N. Langley S.D. Griffin L.D. Elsas L.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 60-64Crossref PubMed Scopus (45) Google Scholar, 7Gronskov K. Vissing H. Shymko R.M. Tornqvist H. DeMeyts P. Biochem. Biophys. Res. Commun. 1993; 192: 905-911Crossref PubMed Scopus (20) Google Scholar, 8Takata Y. Imamura T. Haruta T. Egawa K. Takada Y. Sawa T. Yang G.H. Kobayashi M. Biochem. Biophys. Res. Commun. 1994; 203: 763-772Crossref PubMed Scopus (8) Google Scholar, 9Kadowaki T. Kadowaki H. Accili D. Yazaki Y. Taylor S.I. J. Biol. Chem. 1991; 266: 21224-21231Abstract Full Text PDF PubMed Google Scholar, 10Maassen J.A. Van der Vorm E.R. Van der Zon G.C.M. Klinkhamer M.P. Krans H.M.J. Moller W. Biochemistry. 1991; 30: 10778-10783Crossref PubMed Scopus (43) Google Scholar, 11Accili D. Mosthaf L. Ullrich A. Taylor S.I. J. Biol. Chem. 1991; 266: 434-439Abstract Full Text PDF PubMed Google Scholar, 12van der Vorm E.R. Kuipers A. Kielkopf-Renner S. Krans H.M.J. Moller W. Maassen J.A. J. Biol. Chem. 1994; 269: 14297-14302Abstract Full Text PDF PubMed Google Scholar, 13Cama A. de la Luz Sierra M. Quon M.J. Ottini L. Gorden P. Taylor S.I. J. Biol. Chem. 1993; 268: 8060-8069Abstract Full Text PDF PubMed Google Scholar), and the second is the accelerated intracellular degradation of mutant receptor proteins (14Kadowaki H. Kadowaki T. Cama A. Marcus-Samuels B. Rovira A. Bevins C.L. Taylor S.I. J. Biol. Chem. 1990; 265: 21285-21296Abstract Full Text PDF PubMed Google Scholar, 15Imamura T. Takata Y. Sasaoka T. Takada Y. Morioka H. Haruta T. Sawa T. Iwanishi M. Hu Y.G. Suzuki Y. Hamada J. Kobayashi M. J. Biol. Chem. 1994; 269: 31019-31027Abstract Full Text PDF PubMed Google Scholar). We previously reported the three families of type A insulin-resistant syndrome who had a mutation (Asp1179 or Leu1193) 2The numbering of amino acids in this paper corresponds to the sequence of the receptor of Ebina et al.(2Ebina Y. Ellis L. Jarnagin K. Edery M. Graf L. Clauser E. Ou J.H. Masiarz R. Kan Y.W. Goldfine I.D. Roth R.A. Rutter W.J. Cell. 1985; 40: 747-785Abstract Full Text PDF PubMed Scopus (964) Google Scholar). 2The numbering of amino acids in this paper corresponds to the sequence of the receptor of Ebina et al.(2Ebina Y. Ellis L. Jarnagin K. Edery M. Graf L. Clauser E. Ou J.H. Masiarz R. Kan Y.W. Goldfine I.D. Roth R.A. Rutter W.J. Cell. 1985; 40: 747-785Abstract Full Text PDF PubMed Scopus (964) Google Scholar). in the kinase domain of the insulin receptor leading to the accelerated intracellular degradation of the unprocessed proreceptors (15Imamura T. Takata Y. Sasaoka T. Takada Y. Morioka H. Haruta T. Sawa T. Iwanishi M. Hu Y.G. Suzuki Y. Hamada J. Kobayashi M. J. Biol. Chem. 1994; 269: 31019-31027Abstract Full Text PDF PubMed Google Scholar).Interestingly, Arg209 and Val382 mutant insulin receptors, which were accumulated in the ER, were associated with a molecular chaperone, immunoglobulin heavy chain binding protein (BiP), one of the heat shock proteins in the ER (16Accili D. Kadowaki T. Kadowaki H. Mosthaf L. Ullrich A. Taylor S.I. J. Biol. Chem. 1992; 267: 586-590Abstract Full Text PDF PubMed Google Scholar). Thus, it was likely that the difference in the molecular chaperones to which the mutant insulin receptor tightly bound determined the fate of mutant insulin receptors. Although the association of these specific chaperons with some of mutant insulin receptors has been described, the role of molecular chaperones and the site where these mutants degraded have not been well characterized.The mechanism of intracellular degradation of abnormal proteins in the secretory pathway has not been clearly understood. However, recent observations suggest that unfolded or unassembled proteins are retained in the ER by chaperones, transported back into the cytosol and degraded by the proteasome (17Jentsch S. Schlenker S. Cell. 1995; 82: 881-884Abstract Full Text PDF PubMed Scopus (235) Google Scholar, 18Hiller M.M. Finger A. Schweiger M. Wolf D.H. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (612) Google Scholar, 19Werner E.D. Brodsky J.L. McCracken A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13797-13801Crossref PubMed Scopus (386) Google Scholar, 20Rivett A.J. Biochem. J. 1993; 291: 1-10Crossref PubMed Scopus (380) Google Scholar). In the present study, to investigate the mechanism of the accelerated degradation of both Asp1179and Leu1193 mutant insulin receptors, we examined whether these mutations caused accelerated degradation of unprocessed proreceptors retained in the ER and whether these mutant receptors were degraded by the proteasome, and finally which molecular chaperone was involved in degradation. We now report that the mutant insulin proreceptors in the ER are rapidly transported to the proteasome for degradation through the binding to Hsp90.DISCUSSIONMost of the naturally occurring mutations in the β-subunit of the insulin receptor interfere with processing of proreceptors to the subunits, leading to accumulation of proreceptors in the ER. As reported previously, these mutant insulin receptors were tightly associated with BiP, an intra-ER chaperone, and remained in the ER (16Accili D. Kadowaki T. Kadowaki H. Mosthaf L. Ullrich A. Taylor S.I. J. Biol. Chem. 1992; 267: 586-590Abstract Full Text PDF PubMed Google Scholar). Similarly, the mutant insulin receptor lacking the coding region of exon 13 was not processed to the subunits and was also associated with BiP (22Haruta T. Sawa T. Takata Y. Imamura T. Takada Y. Morioka H. Yang G.H. Kobayashi M. Biochem. J. 1995; 305: 599-604Crossref PubMed Scopus (6) Google Scholar, 29Sawa T. Imamura T. Haruta T. Sasaoka T. Ishiki M. Takata Y. Takada Y. Morioka H. Ishihara H. Usui I. Kobayashi M. Biochem. Biophys. Res. Commun. 1996; 218: 449-453Crossref PubMed Scopus (31) Google Scholar). Since this ΔEx13 insulin receptor remained unprocessed and was easily distinguished from normal matured insulin receptors, we created double mutant insulin receptors that had both ΔEx13 and either Asp1179 or Leu1193 mutations in the tyrosine kinase domain, and monitored the processing and degradation of this mutant insulin receptors. Addition of either Asp1179 or Leu1193 mutations to ΔEx13 insulin receptor led to rapid receptor degradation (15Imamura T. Takata Y. Sasaoka T. Takada Y. Morioka H. Haruta T. Sawa T. Iwanishi M. Hu Y.G. Suzuki Y. Hamada J. Kobayashi M. J. Biol. Chem. 1994; 269: 31019-31027Abstract Full Text PDF PubMed Google Scholar), suggesting that these point mutations were specific for the cause of accelerated degradation of unprocessed insulin receptors that existed in the ER.The ER-associated proteins destined for degradation are transported to the cytosol, where proteolysis is catalyzed by the proteasome (17Jentsch S. Schlenker S. Cell. 1995; 82: 881-884Abstract Full Text PDF PubMed Scopus (235) Google Scholar, 18Hiller M.M. Finger A. Schweiger M. Wolf D.H. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (612) Google Scholar). By using two proteasome inhibitors, we clearly demonstrated that proteasome played a major role in degradation of the mutant receptors. Furthermore, the inhibitors increased the amount of insulin receptor protein in the transformed lymphocyte derived from the patient with Asp1179 mutation. Thus, the proteasome inhibitors rescued these mutant receptors, which could be transported to the cell surface. These results suggest that both Asp1179 and Leu1193 mutant insulin proreceptors are degraded by the proteasome in the cytosol before their transport from the ER to the Golgi apparatus, because the degradation occurs before the processing from the proreceptor to the α- and β-subunits takes place at the Golgi apparatus.The degradation by the proteasome usually requires polyubiquitination of the targeted proteins (18Hiller M.M. Finger A. Schweiger M. Wolf D.H. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (612) Google Scholar, 30Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1127) Google Scholar, 31Biederer T. Volkwein C. Sommer T. EMBO J. 1996; 15: 2069-2076Crossref PubMed Scopus (239) Google Scholar, 32Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). However, we could not detect any specific association of ubiquitin with these mutant insulin receptors. Therefore, Asp1179 and Leu1193 mutant insulin receptor proteins were degraded by the proteasome without direct ubiquitination in the transfected COS-7 cells and the patient's cells. In fact, the proteasome degradation of certain proteins does not require ubiquitination (19Werner E.D. Brodsky J.L. McCracken A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13797-13801Crossref PubMed Scopus (386) Google Scholar, 33Wiertz E.J.H.J. Tortorella D. Bogyo M. Yu J. Mothes W. Jones T.R. Rapoport T.A. Ploegh H.L. Nature. 1996; 384: 432-438Crossref PubMed Scopus (949) Google Scholar, 34McGee T.P. Cheng H.H. Kumagai H. Omura S. Simoni R.D. J. Biol. Chem. 1996; 271: 25630-25638Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar).To determine the mechanism by which these mutant insulin proreceptors were transported out of the ER and then presented to the proteasome, we tested the possibility that specific molecular chaperones might bind to the mutant proteins and present them to the proteasome. We demonstrated that Hsp90 was tightly associated with these mutant receptors, both in the cells and in the cell-free system, using GST fusion proteins. Furthermore, the degradation of the mutant insulin receptor protein was partially inhibited by microinjection of the anti-Hsp90 antibody, leading to the increased amount of these mutant insulin receptors in the cells. These experiments suggested that Hsp90 played a key role in transport of these mutant receptors out of the ER and in the subsequent degradation by the proteasome in the cytosol.Hsp90, which exists in the cytosol, functions as a molecular chaperon, by the complex formation with steroid receptor (35Bohen S.P. Yamamoto K.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11424-11428Crossref PubMed Scopus (192) Google Scholar, 36Nathan D. Lindquist S. Mol. Cell. Biol. 1995; 15: 3917-3925Crossref PubMed Scopus (368) Google Scholar), pp60v-src (37Xu Y. Lindquist S.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7074-7078Crossref PubMed Scopus (377) Google Scholar,38Rutherford S. Zucker C. Cell. 1994; 79: 1129-1132Abstract Full Text PDF PubMed Scopus (169) Google Scholar), Raf-1 (39Stancato L.F. Chow Y.-H. Huchinson K.A. Perdew G.H. Jove R. Pratt W.B. J. Biol. Chem. 1993; 268: 21711-21716Abstract Full Text PDF PubMed Google Scholar), or casein kinase II (40Miyata Y. Yahara I. Biochemistry. 1995; 34: 8123-8129Crossref PubMed Scopus (103) Google Scholar). However, it has not been reported that Hsp90 participates in the degradation of ER-associated misfolded proteins. Hsp90 functions in co-operation with several kinds of molecular chaperones, such as Hsc70, as we demonstrated in GST-IRc-Hsp90 binding studies in the presence of Hsc70. Furthermore, we observed that Hsp90 associated with ubiquitin in the COS-7 cells expressing Asp1179 or Leu1193 mutant type or wild-type insulin receptors. These results suggest that the association of the mutant insulin receptors with Hsp90 might be followed by further complex formation with ubiquitin and Hsc70. It is also possible that other chaperons, which we could not identify, might participate in the ER-associated protein degradation.The mechanism whereby misfolded proteins are dislocated from the ER is not clearly understood. Recent observations by Weirtz et al.(33Wiertz E.J.H.J. Tortorella D. Bogyo M. Yu J. Mothes W. Jones T.R. Rapoport T.A. Ploegh H.L. Nature. 1996; 384: 432-438Crossref PubMed Scopus (949) Google Scholar) suggest that the Sec61 complex is thought to have a role in the dislocation of proteins from the ER to the cytosol, and that the misfolded proteins could be caught in transit through the channel. It should be further investigated whether the mutant insulin receptors such as Asp1179 or Leu1193 mutant receptor are also dislocated from the ER to the cytosol in the similar fashion.Asp1179 and Leu1193 mutations may cause a significant change in tertiary structure of the receptor that can be specifically recognized by Hsp90. The close location and surface exposure of Glu1179 and Trp1193 in the tertiary structure of insulin receptor β-subunits (41Hubbard S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (954) Google Scholar) would support the importance of these amino acids for Hsp90 binding. Thus, it is likely that other mutations in the kinase domain may cause similar phenomena as shown in Asp1179 and Leu1193 mutations. Another mutation, Asp1048 in the kinase domain (42Haruta T. Takata Y. Iwanishi M. Maegawa H. Imamura T. Egawa K. Itazu T. Kobayashi M. Diabetes. 1993; 42: 1837-1844Crossref PubMed Google Scholar), however, did not lead to accelerated degradation, probably due to a relatively normally folded structure of the receptor protein. Although there are some other mutants of the kinase domain that show the decreased insulin binding (43Cama A. Quon M.J. de la Luz Sierra M. Taylor S.I. J. Biol. Chem. 1992; 267: 8383-8389Abstract Full Text PDF PubMed Google Scholar, 44Moller D.E. Yokota A. Ginsberg-Fellner F. Flier J.S. Mol. Endocrinol. 1990; 4: 1183-1191Crossref PubMed Scopus (49) Google Scholar), it remains to be determined whether they are also degraded in the same manner as described above. On the other hand, in the α-subunit of the insulin receptor, only the Glu460 mutation showed the accelerated receptor degradation (14Kadowaki H. Kadowaki T. Cama A. Marcus-Samuels B. Rovira A. Bevins C.L. Taylor S.I. J. Biol. Chem. 1990; 265: 21285-21296Abstract Full Text PDF PubMed Google Scholar). Since it was reported that the Glu460 mutation caused lysosomal degradation of the insulin receptor on the way of recycling pathway, the mechanism of this degradation was different from that of Asp1179 and Leu1193 mutant receptors.In conclusion, we have clarified a novel mechanism for degradation of the mutant insulin receptors by proteasome, in which Hsp90 plays a key role. Various mutations in the insulin receptor gene have been reported in patients with severe insulin resistance (1Taylor S.I. Diabetes. 1992; 41: 1473-1490Crossref PubMed Scopus (0) Google Scholar). Analysis of the mutated insulin receptors in the cells gave us the opportunity to understand the function and processing of insulin receptor protein. Among the various mechanisms for the insulin resistance in these patients, certain patients with the insulin receptor mutations showed a reduced number of insulin receptors on the cell surface although the mRNA of insulin receptor was normally expressed (1Taylor S.I. Diabetes. 1992; 41: 1473-1490Crossref PubMed Scopus (0) Google Scholar). Two major causes for this phenomenon have been described; the first is the impaired protein processing of mutated insulin receptors and accumulation in the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; Hsp, heat shock protein; Hsc, heat shock cognate; BiP, immunoglobulin heavy chain binding protein; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; IRc, cytoplasmic domain of insulin receptor. 1The abbreviations used are: ER, endoplasmic reticulum; Hsp, heat shock protein; Hsc, heat shock cognate; BiP, immunoglobulin heavy chain binding protein; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; IRc, cytoplasmic domain of insulin receptor.(3Kadowaki T. Kadowaki H. Accili D. Taylor S.I. J. Biol. Chem. 1990; 265: 19143-19150Abstract Full Text PDF PubMed Google Scholar, 4Wertheimer E. Barbetti F. Muggeo M. Roth J. Taylor S.I. J. Biol. Chem. 1994; 269: 7587-7592Abstract Full Text PDF PubMed Google Scholar, 5Van der Vorm E.R. Van der Zon G.C.M. Moller W. Krans H.M.J. Lindhout D. Maassen J.A. J. Biol. Chem. 1992; 267: 66-71Abstract Full Text PDF PubMed Google Scholar, 6Longo N. Langley S.D. Griffin L.D. Elsas L.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 60-64Crossref PubMed Scopus (45) Google Scholar, 7Gronskov K. Vissing H. Shymko R.M. Tornqvist H. DeMeyts P. Biochem. Biophys. Res. Commun. 1993; 192: 905-911Crossref PubMed Scopus (20) Google Scholar, 8Takata Y. Imamura T. Haruta T. Egawa K. Takada Y. Sawa T. Yang G.H. Kobayashi M. Biochem. Biophys. Res. Commun. 1994; 203: 763-772Crossref PubMed Scopus (8) Google Scholar, 9Kadowaki T. Kadowaki H. Accili D. Yazaki Y. Taylor S.I. J. Biol. Chem. 1991; 266: 21224-21231Abstract Full Text PDF PubMed Google Scholar, 10Maassen J.A. Van der Vorm E.R. Van der Zon G.C.M. Klinkhamer M.P. Krans H.M.J. Moller W. Biochemistry. 1991; 30: 10778-10783Crossref PubMed Scopus (43) Google Scholar, 11Accili D. Mosthaf L. Ullrich A. Taylor S.I. J. Biol. Chem. 1991; 266: 434-439Abstract Full Text PDF PubMed Google Scholar, 12van der Vorm E.R. Kuipers A. Kielkopf-Renner S. Krans H.M.J. Moller W. Maassen J.A. J. Biol. Chem. 1994; 269: 14297-14302Abstract Full Text PDF PubMed Google Scholar, 13Cama A. de la Luz Sierra M. Quon M.J. Ottini L. Gorden P. Taylor S.I. J. Biol. Chem. 1993; 268: 8060-8069Abstract Full Text PDF PubMed Google Scholar), and the second is the accelerated intracellular degradation of mutant receptor proteins (14Kadowaki H. Kadowaki T. Cama A. Marcus-Samuels B. Rovira A. Bevins C.L. Taylor S.I. J. Biol. Chem. 1990; 265: 21285-21296Abstract Full Text PDF PubMed Google Scholar, 15Imamura T. Takata Y. Sasaoka T. Takada Y. Morioka H. Haruta T. Sawa T. Iwanishi M. Hu Y.G. Suzuki Y. Hamada J. Kobayashi M. J. Biol. Chem. 1994; 269: 31019-31027Abstract Full Text PDF PubMed Google Scholar). We previously reported the three families of type A insulin-resistant syndrome who had a mutation (Asp1179 or Leu1193) 2The numbering of amino acids in this paper corresponds to the sequence of the receptor of Ebina et al.(2Ebina Y. Ellis L. Jarnagin K. Edery M. Graf L. Clauser E. Ou J.H. Masiarz R. Kan Y.W. Goldfine I.D. Roth R.A. Rutter W.J. Cell. 1985; 40: 747-785Abstract Full Text PDF PubMed Scopus (964) Google Scholar). 2The numbering of amino acids in this paper corresponds to the sequence of the receptor of Ebina et al.(2Ebina Y. Ellis L. Jarnagin K. Edery M. Graf L. Clauser E. Ou J.H. Masiarz R. Kan Y.W. Goldfine I.D. Roth R.A. Rutter W.J. Cell. 1985; 40: 747-785Abstract Full Text PDF PubMed Scopus (964) Google Scholar). in the kinase domain of the insulin receptor leading to the accelerated intracellular degradation of the unprocessed proreceptors (15Imamura T. Takata Y. Sasaoka T. Takada Y. Morioka H. Haruta T. Sawa T. Iwanishi M. Hu Y.G. Suzuki Y. Hamada J. Kobayashi M. J. Biol. Chem. 1994; 269: 31019-31027Abstract Full Text PDF PubMed Google Scholar). Interestingly, Arg209 and Val382 mutant insulin receptors, which were accumulated in the ER, were associated with a molecular chaperone, immunoglobulin heavy chain binding protein (BiP), one of the heat shock proteins in the ER (16Accili D. Kadowaki T. Kadowaki H. Mosthaf L. Ullrich A. Taylor S.I. J. Biol. Chem. 1992; 267: 586-590Abstract Full Text PDF PubMed Google Scholar). Thus, it was likely that the difference in the molecular chaperones to which the mutant insulin receptor tightly bound determined the fate of mutant insulin receptors. Although the association of these specific chaperons with some of mutant insulin receptors has been described, the role of molecular chaperones and the site where these mutants degraded have not been well characterized. The mechanism of intracellular degradation of abnormal proteins in the secretory pathway has not been clearly understood. However, recent observations suggest that unfolded or unassembled proteins are retained in the ER by chaperones, transported back into the cytosol and degraded by the proteasome (17Jentsch S. Schlenker S. Cell. 1995; 82: 881-884Abstract Full Text PDF PubMed Scopus (235) Google Scholar, 18Hiller M.M. Finger A. Schweiger M. Wolf D.H. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (612) Google Scholar, 19Werner E.D. Brodsky J.L. McCracken A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13797-13801Crossref PubMed Scopus (386) Google Scholar, 20Rivett A.J. Biochem. J. 1993; 291: 1-10Crossref PubMed Scopus (380) Google Scholar). In the present study, to investigate the mechanism of the accelerated degradation of both Asp1179and Leu1193 mutant insulin receptors, we examined whether these mutations caused accelerated degradation of unprocessed proreceptors retained in the ER and whether these mutant receptors were degraded by the proteasome, and finally which molecular chaperone was involved in degradation. We now report that the mutant insulin proreceptors in the ER are rapidly transported to the proteasome for degradation through the binding to Hsp90. DISCUSSIONMost of the naturally occurring mutations in the β-subunit of the insulin receptor interfere with processing of proreceptors to the subunits, leading to accumulation of proreceptors in the ER. As reported previously, these mutant insulin receptors were tightly associated with BiP, an intra-ER chaperone, and remained in the ER (16Accili D. Kadowaki T. Kadowaki H. Mosthaf L. Ullrich A. Taylor S.I. J. Biol. Chem. 1992; 267: 586-590Abstract Full Text PDF PubMed Google Scholar). Similarly, the mutant insulin receptor lacking the coding region of exon 13 was not processed to the subunits and was also associated with BiP (22Haruta T. Sawa T. Takata Y. Imamura T. Takada Y. Morioka H. Yang G.H. Kobayashi M. Biochem. J. 1995; 305: 599-604Crossref PubMed Scopus (6) Google Scholar, 29Sawa T. Imamura T. Haruta T. Sasaoka T. Ishiki M. Takata Y. Takada Y. Morioka H. Ishihara H. Usui I. Kobayashi M. Biochem. Biophys. Res. Commun. 1996; 218: 449-453Crossref PubMed Scopus (31) Google Scholar). Since this ΔEx13 insulin receptor remained unprocessed and was easily distinguished from normal matured insulin receptors, we created double mutant insulin receptors that had both ΔEx13 and either Asp1179 or Leu1193 mutations in the tyrosine kinase domain, and monitored the processing and degradation of this mutant insulin receptors. Addition of either Asp1179 or Leu1193 mutations to ΔEx13 insulin receptor led to rapid receptor degradation (15Imamura T. Takata Y. Sasaoka T. Takada Y. Morioka H. Haruta T. Sawa T. Iwanishi M. Hu Y.G. Suzuki Y. Hamada J. Kobayashi M. J. Biol. Chem. 1994; 269: 31019-31027Abstract Full Text PDF PubMed Google Scholar), suggesting that these point mutations were specific for the cause of accelerated degradation of unprocessed insulin receptors that existed in the ER.The ER-associated proteins destined for degradation are transported to the cytosol, where proteolysis is catalyzed by the proteasome (17Jentsch S. Schlenker S. Cell. 1995; 82: 881-884Abstract Full Text PDF PubMed Scopus (235) Google Scholar, 18Hiller M.M. Finger A. Schweiger M. Wolf D.H. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (612) Google Scholar). By using two proteasome inhibitors, we clearly demonstrated that proteasome played a major role in degradation of the mutant receptors. Furthermore, the inhibitors increased the amount of insulin receptor protein in the transformed lymphocyte derived from the patient with Asp1179 mutation. Thus, the proteasome inhibitors rescued these mutant receptors, which could be transported to the cell surface. These results suggest that both Asp1179 and Leu1193 mutant insulin proreceptors are degraded by the proteasome in the cytosol before their transport from the ER to the Golgi apparatus, because the degradation occurs before the processing from the proreceptor to the α- and β-subunits takes place at the Golgi apparatus.The degradation by the proteasome usually requires polyubiquitination of the targeted proteins (18Hiller M.M. Finger A. Schweiger M. Wolf D.H. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (612) Google Scholar, 30Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1127) Google Scholar, 31Biederer T. Volkwein C. Sommer T. EMBO J. 1996; 15: 2069-2076Crossref PubMed Scopus (239) Google Scholar, 32Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). However, we could not detect any specific association of ubiquitin with these mutant insulin receptors. Therefore, Asp1179 and Leu1193 mutant insulin receptor proteins were degraded by the proteasome without direct ubiquitination in the transfected COS-7 cells and the patient's cells. In fact, the proteasome degradation of certain proteins does not require ubiquitination (19Werner E.D. Brodsky J.L. McCracken A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13797-13801Crossref PubMed Scopus (386) Google Scholar, 33Wiertz E.J.H.J. Tortorella D. Bogyo M. Yu J. Mothes W. Jones T.R. Rapoport T.A. Ploegh H.L. Nature. 1996; 384: 432-438Crossref PubMed Scopus (949) Google Scholar, 34McGee T.P. Cheng H.H. Kumagai H. Omura S. Simoni R.D. J. Biol. Chem. 1996; 271: 25630-25638Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar).To determine the mechanism by which these mutant insulin proreceptors were transported out of the ER and then presented to the proteasome, we tested the possibility that specific molecular chaperones might bind to the mutant proteins and present them to the proteasome. We demonstrated that Hsp90 was tightly associated with these mutant receptors, both in the cells and in the cell-free system, using GST fusion proteins. Furthermore, the degradation of the mutant insulin receptor protein was partially inhibited by microinjection of the anti-Hsp90 antibody, leading to the increased amount of these mutant insulin receptors in the cells. These experiments suggested that Hsp90 played a key role in transport of these mutant receptors out of the ER and in the subsequent degradation by the proteasome in the cytosol.Hsp90, which exists in the cytosol, functions as a molecular chaperon, by the complex formation with steroid receptor (35Bohen S.P. Yamamoto K.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11424-11428Crossref PubMed Scopus (192) Google Scholar, 36Nathan D. Lindquist S. Mol. Cell. Biol. 1995; 15: 3917-3925Crossref PubMed Scopus (368) Google Scholar), pp60v-src (37Xu Y. Lindquist S.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7074-7078Crossref PubMed Scopus (377) Google Scholar,38Rutherford S. Zucker C. Cell. 1994; 79: 1129-1132Abstract Full Text PDF PubMed Scopus (169) Google Scholar), Raf-1 (39Stancato L.F. Chow Y.-H. Huchinson K.A. Perdew G.H. Jove R. Pratt W.B. J. Biol. Chem. 1993; 268: 21711-21716Abstract Full Text PDF PubMed Google Scholar), or casein kinase II (40Miyata Y. Yahara I. Biochemistry. 1995; 34: 8123-8129Crossref PubMed Scopus (103) Google Scholar). However, it has not been reported that Hsp90 participates in the degradation of ER-associated misfolded proteins. Hsp90 functions in co-operation with several kinds of molecular chaperones, such as Hsc70, as we demonstrated in GST-IRc-Hsp90 binding studies in the presence of Hsc70. Furthermore, we observed that Hsp90 associated with ubiquitin in the COS-7 cells expressing Asp1179 or Leu1193 mutant type or wild-type insulin receptors. These results suggest that the association of the mutant insulin receptors with Hsp90 might be followed by further complex formation with ubiquitin and Hsc70. It is also possible that other chaperons, which we could not identify, might participate in the ER-associated protein degradation.The mechanism whereby misfolded proteins are dislocated from the ER is not clearly understood. Recent observations by Weirtz et al.(33Wiertz E.J.H.J. Tortorella D. Bogyo M. Yu J. Mothes W. Jones T.R. Rapoport T.A. Ploegh H.L. Nature. 1996; 384: 432-438Crossref PubMed Scopus (949) Google Scholar) suggest that the Sec61 complex is thought to have a role in the dislocation of proteins from the ER to the cytosol, and that the misfolded proteins could be caught in transit through the channel. It should be further investigated whether the mutant insulin receptors such as Asp1179 or Leu1193 mutant receptor are also dislocated from the ER to the cytosol in the similar fashion.Asp1179 and Leu1193 mutations may cause a significant change in tertiary structure of the receptor that can be specifically recognized by Hsp90. The close location and surface exposure of Glu1179 and Trp1193 in the tertiary structure of insulin receptor β-subunits (41Hubbard S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (954) Google Scholar) would support the importance of these amino acids for Hsp90 binding. Thus, it is likely that other mutations in the kinase domain may cause similar phenomena as shown in Asp1179 and Leu1193 mutations. Another mutation, Asp1048 in the kinase domain (42Haruta T. Takata Y. Iwanishi M. Maegawa H. Imamura T. Egawa K. Itazu T. Kobayashi M. Diabetes. 1993; 42: 1837-1844Crossref PubMed Google Scholar), however, did not lead to accelerated degradation, probably due to a relatively normally folded structure of the receptor protein. Although there are some other mutants of the kinase domain that show the decreased insulin binding (43Cama A. Quon M.J. de la Luz Sierra M. Taylor S.I. J. Biol. Chem. 1992; 267: 8383-8389Abstract Full Text PDF PubMed Google Scholar, 44Moller D.E. Yokota A. Ginsberg-Fellner F. Flier J.S. Mol. Endocrinol. 1990; 4: 1183-1191Crossref PubMed Scopus (49) Google Scholar), it remains to be determined whether they are also degraded in the same manner as described above. On the other hand, in the α-subunit of the insulin receptor, only the Glu460 mutation showed the accelerated receptor degradation (14Kadowaki H. Kadowaki T. Cama A. Marcus-Samuels B. Rovira A. Bevins C.L. Taylor S.I. J. Biol. Chem. 1990; 265: 21285-21296Abstract Full Text PDF PubMed Google Scholar). Since it was reported that the Glu460 mutation caused lysosomal degradation of the insulin receptor on the way of recycling pathway, the mechanism of this degradation was different from that of Asp1179 and Leu1193 mutant receptors.In conclusion, we have clarified a novel mechanism for degradation of the mutant insulin receptors by proteasome, in which Hsp90 plays a key role. Most of the naturally occurring mutations in the β-subunit of the insulin receptor interfere with processing of proreceptors to the subunits, leading to accumulation of proreceptors in the ER. As reported previously, these mutant insulin receptors were tightly associated with BiP, an intra-ER chaperone, and remained in the ER (16Accili D. Kadowaki T. Kadowaki H. Mosthaf L. Ullrich A. Taylor S.I. J. Biol. Chem. 1992; 267: 586-590Abstract Full Text PDF PubMed Google Scholar). Similarly, the mutant insulin receptor lacking the coding region of exon 13 was not processed to the subunits and was also associated with BiP (22Haruta T. Sawa T. Takata Y. Imamura T. Takada Y. Morioka H. Yang G.H. Kobayashi M. Biochem. J. 1995; 305: 599-604Crossref PubMed Scopus (6) Google Scholar, 29Sawa T. Imamura T. Haruta T. Sasaoka T. Ishiki M. Takata Y. Takada Y. Morioka H. Ishihara H. Usui I. Kobayashi M. Biochem. Biophys. Res. Commun. 1996; 218: 449-453Crossref PubMed Scopus (31) Google Scholar). Since this ΔEx13 insulin receptor remained unprocessed and was easily distinguished from normal matured insulin receptors, we created double mutant insulin receptors that had both ΔEx13 and either Asp1179 or Leu1193 mutations in the tyrosine kinase domain, and monitored the processing and degradation of this mutant insulin receptors. Addition of either Asp1179 or Leu1193 mutations to ΔEx13 insulin receptor led to rapid receptor degradation (15Imamura T. Takata Y. Sasaoka T. Takada Y. Morioka H. Haruta T. Sawa T. Iwanishi M. Hu Y.G. Suzuki Y. Hamada J. Kobayashi M. J. Biol. Chem. 1994; 269: 31019-31027Abstract Full Text PDF PubMed Google Scholar), suggesting that these point mutations were specific for the cause of accelerated degradation of unprocessed insulin receptors that existed in the ER. The ER-associated proteins destined for degradation are transported to the cytosol, where proteolysis is catalyzed by the proteasome (17Jentsch S. Schlenker S. Cell. 1995; 82: 881-884Abstract Full Text PDF PubMed Scopus (235) Google Scholar, 18Hiller M.M. Finger A. Schweiger M. Wolf D.H. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (612) Google Scholar). By using two proteasome inhibitors, we clearly demonstrated that proteasome played a major role in degradation of the mutant receptors. Furthermore, the inhibitors increased the amount of insulin receptor protein in the transformed lymphocyte derived from the patient with Asp1179 mutation. Thus, the proteasome inhibitors rescued these mutant receptors, which could be transported to the cell surface. These results suggest that both Asp1179 and Leu1193 mutant insulin proreceptors are degraded by the proteasome in the cytosol before their transport from the ER to the Golgi apparatus, because the degradation occurs before the processing from the proreceptor to the α- and β-subunits takes place at the Golgi apparatus. The degradation by the proteasome usually requires polyubiquitination of the targeted proteins (18Hiller M.M. Finger A. Schweiger M. Wolf D.H. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (612) Google Scholar, 30Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1127) Google Scholar, 31Biederer T. Volkwein C. Sommer T. EMBO J. 1996; 15: 2069-2076Crossref PubMed Scopus (239) Google Scholar, 32Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). However, we could not detect any specific association of ubiquitin with these mutant insulin receptors. Therefore, Asp1179 and Leu1193 mutant insulin receptor proteins were degraded by the proteasome without direct ubiquitination in the transfected COS-7 cells and the patient's cells. In fact, the proteasome degradation of certain proteins does not require ubiquitination (19Werner E.D. Brodsky J.L. McCracken A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13797-13801Crossref PubMed Scopus (386) Google Scholar, 33Wiertz E.J.H.J. Tortorella D. Bogyo M. Yu J. Mothes W. Jones T.R. Rapoport T.A. Ploegh H.L. Nature. 1996; 384: 432-438Crossref PubMed Scopus (949) Google Scholar, 34McGee T.P. Cheng H.H. Kumagai H. Omura S. Simoni R.D. J. Biol. Chem. 1996; 271: 25630-25638Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). To determine the mechanism by which these mutant insulin proreceptors were transported out of the ER and then presented to the proteasome, we tested the possibility that specific molecular chaperones might bind to the mutant proteins and present them to the proteasome. We demonstrated that Hsp90 was tightly associated with these mutant receptors, both in the cells and in the cell-free system, using GST fusion proteins. Furthermore, the degradation of the mutant insulin receptor protein was partially inhibited by microinjection of the anti-Hsp90 antibody, leading to the increased amount of these mutant insulin receptors in the cells. These experiments suggested that Hsp90 played a key role in transport of these mutant receptors out of the ER and in the subsequent degradation by the proteasome in the cytosol. Hsp90, which exists in the cytosol, functions as a molecular chaperon, by the complex formation with steroid receptor (35Bohen S.P. Yamamoto K.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11424-11428Crossref PubMed Scopus (192) Google Scholar, 36Nathan D. Lindquist S. Mol. Cell. Biol. 1995; 15: 3917-3925Crossref PubMed Scopus (368) Google Scholar), pp60v-src (37Xu Y. Lindquist S.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7074-7078Crossref PubMed Scopus (377) Google Scholar,38Rutherford S. Zucker C. Cell. 1994; 79: 1129-1132Abstract Full Text PDF PubMed Scopus (169) Google Scholar), Raf-1 (39Stancato L.F. Chow Y.-H. Huchinson K.A. Perdew G.H. Jove R. Pratt W.B. J. Biol. Chem. 1993; 268: 21711-21716Abstract Full Text PDF PubMed Google Scholar), or casein kinase II (40Miyata Y. Yahara I. Biochemistry. 1995; 34: 8123-8129Crossref PubMed Scopus (103) Google Scholar). However, it has not been reported that Hsp90 participates in the degradation of ER-associated misfolded proteins. Hsp90 functions in co-operation with several kinds of molecular chaperones, such as Hsc70, as we demonstrated in GST-IRc-Hsp90 binding studies in the presence of Hsc70. Furthermore, we observed that Hsp90 associated with ubiquitin in the COS-7 cells expressing Asp1179 or Leu1193 mutant type or wild-type insulin receptors. These results suggest that the association of the mutant insulin receptors with Hsp90 might be followed by further complex formation with ubiquitin and Hsc70. It is also possible that other chaperons, which we could not identify, might participate in the ER-associated protein degradation. The mechanism whereby misfolded proteins are dislocated from the ER is not clearly understood. Recent observations by Weirtz et al.(33Wiertz E.J.H.J. Tortorella D. Bogyo M. Yu J. Mothes W. Jones T.R. Rapoport T.A. Ploegh H.L. Nature. 1996; 384: 432-438Crossref PubMed Scopus (949) Google Scholar) suggest that the Sec61 complex is thought to have a role in the dislocation of proteins from the ER to the cytosol, and that the misfolded proteins could be caught in transit through the channel. It should be further investigated whether the mutant insulin receptors such as Asp1179 or Leu1193 mutant receptor are also dislocated from the ER to the cytosol in the similar fashion. Asp1179 and Leu1193 mutations may cause a significant change in tertiary structure of the receptor that can be specifically recognized by Hsp90. The close location and surface exposure of Glu1179 and Trp1193 in the tertiary structure of insulin receptor β-subunits (41Hubbard S.R. Wei L. Ellis L. Hendrickson W.A. Nature. 1994; 372: 746-754Crossref PubMed Scopus (954) Google Scholar) would support the importance of these amino acids for Hsp90 binding. Thus, it is likely that other mutations in the kinase domain may cause similar phenomena as shown in Asp1179 and Leu1193 mutations. Another mutation, Asp1048 in the kinase domain (42Haruta T. Takata Y. Iwanishi M. Maegawa H. Imamura T. Egawa K. Itazu T. Kobayashi M. Diabetes. 1993; 42: 1837-1844Crossref PubMed Google Scholar), however, did not lead to accelerated degradation, probably due to a relatively normally folded structure of the receptor protein. Although there are some other mutants of the kinase domain that show the decreased insulin binding (43Cama A. Quon M.J. de la Luz Sierra M. Taylor S.I. J. Biol. Chem. 1992; 267: 8383-8389Abstract Full Text PDF PubMed Google Scholar, 44Moller D.E. Yokota A. Ginsberg-Fellner F. Flier J.S. Mol. Endocrinol. 1990; 4: 1183-1191Crossref PubMed Scopus (49) Google Scholar), it remains to be determined whether they are also degraded in the same manner as described above. On the other hand, in the α-subunit of the insulin receptor, only the Glu460 mutation showed the accelerated receptor degradation (14Kadowaki H. Kadowaki T. Cama A. Marcus-Samuels B. Rovira A. Bevins C.L. Taylor S.I. J. Biol. Chem. 1990; 265: 21285-21296Abstract Full Text PDF PubMed Google Scholar). Since it was reported that the Glu460 mutation caused lysosomal degradation of the insulin receptor on the way of recycling pathway, the mechanism of this degradation was different from that of Asp1179 and Leu1193 mutant receptors. In conclusion, we have clarified a novel mechanism for degradation of the mutant insulin receptors by proteasome, in which Hsp90 plays a key role. We thank Prof. Yukio Ikehara (Department of Biochemistry, Fukuoka University School of Medicine, Japan) for helpful discussion and critical review of the manuscript.