From the First Department of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan
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ABSTRACT |
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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 (Ex13-IR), which
were accumulated in the endoplasmic reticulum as unprocessed
proreceptors. The addition of Asp1179 or
Leu1193 mutation to
Ex13-IR caused accelerated
degradation of the unprocessed
Ex13-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.
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INTRODUCTION |
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Various mutations in the insulin receptor gene have been reported in patients with severe insulin resistance (1). 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 (1). 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)1 (3-13), and the second is the accelerated intracellular degradation of mutant receptor proteins (14, 15). We previously reported the three families of type A insulin-resistant syndrome who had a mutation (Asp1179 or Leu1193)2 in the kinase domain of the insulin receptor leading to the accelerated intracellular degradation of the unprocessed proreceptors (15).
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 (16). 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 (17-20). In the present study, to investigate the mechanism of the accelerated degradation of both Asp1179 and 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.
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EXPERIMENTAL PROCEDURES |
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Cell Lines and Materials--
COS-7 cells were maintained in
Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf
serum as described (21). GST gene fusion system was purchased from
Amersham Pharmacia Biotech. Proteasome inhibitors MG-115 and MG-132 and
protease inhibitor E-64-d were from Peptide Institute Inc. (Osaka,
Japan). Enhanced chemiluminescence reagents were from Amersham
Pharmacia Biotech. Hsp90 and Hsc70 protein, and antibodies against
Hsp90, Hsc70, Hsp60, BiP, and ubiquitin were purchased from StressGen
(Victoria, Canada). Anti-calnexin antibodies were kindly provided by
Dr. I. Wada (Sapporo University of Medical Science, Sapporo, Japan), and polyclonal anti-insulin receptor C-terminal antibody was provided by Dr. J. M. Olefsky. Mouse monoclonal anti-insulin receptor
-subunit antibody was purchased from Cosmo Bio Co. Ltd. (Tokyo,
Japan). Mouse immunoglobulin G (IgG) and fluorescein isothiocyanate
(FITC)- or rhodamine-conjugated anti-rabbit and anti-mouse IgG
antibodies were from Jackson Laboratories (West Grove, NY).
Electrophoresis reagents were from Bio-Rad. All other reagents were of
analytical grade and were purchased from Sigma or Wako Pure Chemical
Industries (Osaka, Japan).
Construction and Expression of Wild-type and Mutant Insulin
Receptors--
Expression plasmids (pGEM3SVHIR) of wild-type and
mutant (exon 13 deletion (Ex13), Asp1179,
Leu1193,
Ex13+Asp1179, and
Ex13+Leu1193) human insulin receptor were constructed as
described previously (15, 22). COS-7 cells were transfected with
these vectors by electroporation, using a Gene Pulser (Bio-Rad). The
cells were electroporated with a total of 40 µg of DNA at 340 V and
960 microfarads to obtain a high degree of transfection efficiency,
according to Yamauchi et al. (23).
Biosynthetic Labeling-- COS-7 cells were used for steady-state labeling experiments at 60 h after transfection. The transfected cells were incubated in methionine-free DMEM containing 10% dialyzed fetal bovine serum and Tran35S-label (0.1 mCi/ml; 1022 Ci/mmol; ICN Biochemicals Inc.) at 37 °C for 12 h, followed by incubation in complete DMEM for 21 h. The cells were solubilized in a buffer containing 50 mM HEPES (pH 7.6), 10 mM MgSO4, 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, and 1 mg/ml aprotinin, and the cell lysate was applied to a wheat germ agglutinin-agarose column. The eluate was immunoprecipitated with anti-insulin receptor antibody and analyzed by SDS-PAGE and autoradiography (15).
Expression of Glutathione S-Transferase-Insulin Receptor Cytoplasmic Domain Fusion Protein-- The cytoplasmic domain cDNAs of the wild-type and mutant insulin receptors were digested from pGEM3SVHIR expression vectors with BglI (codon 958) and SpeI (nucleotide 13100 of 3' intron) (24). The fragments containing the cytoplasmic domain of insulin receptor (IRc) were filled into blunt end by use of T4 DNA polymerase. The GST fusion protein expression vector pGEX-5X-2 was digested with SmaI, filled into blunt end similarly, and treated with calf intestine alkaline phosphatase. These two fragments were ligated, and then resulting plasmids were transfected into Escherichia coli JM109-competent cells (Takara, Tokyo, Japan). The plasmids containing the cytoplasmic insulin receptor cDNA were selected by enzyme restriction and confirmed by sequencing. After transfection of the plasmids containing GST-IRc cDNA into E. coli BL21 strain by electroporation, GST-IRc fusion proteins were purified according to the specifications of the manufacturer (Amersham Pharmacia Biotech).
Association of the GST-IRc Fusion Proteins with Heat Shock Proteins-- After purification through glutathione-Sepharose 4B, 3 µg each of the fusion proteins were incubated with 9.9 µg of Hsp90 and/or Hsc70 in 1 ml of phosphate-buffered saline (PBS) for 1 h at 4 °C. After washing three times with PBS, the glutathione-Sepharose 4B-coupled protein complex was centrifuged at 500 × g and was dissolved in Laemmli sample buffer (25) to be analyzed by Western blotting.
Western Blotting Analysis-- Cells were washed three times with PBS and solubilized in boiled Laemmli sample buffer for experiments of the proteasome inhibitors. For the immunoprecipitation assay, cells were lysed in a pH 7.4 buffer, containing 30 mM Tris, 150 mM NaCl, 10 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. The cell lysates were centrifuged to remove insoluble materials. The supernatants were used for immunoprecipitation with the indicated antibodies for 4 h at 4 °C. The precipitates were separated by SDS-PAGE and transferred to Immobilon-P using a Bio-Rad Transblot apparatus. The membranes were blocked in a pH 7.5 buffer, containing 50 mM Tris, 150 mM NaCl, 0.1% Tween 20, and 2.5% bovine serum albumin, for 2 h at 20 °C. The membranes were then probed with specified antibodies for 2 h at 20 °C. After washing the membranes in a pH 7.5 buffer, containing 50 mM Tris, 150 mM NaCl, and 0.1% Tween 20, blots were incubated with horseradish peroxidase-linked second antibody followed by enhanced chemiluminescence detection using the ECL reagent according to the manufacturer's instructions (Amersham Pharmacia Biotech) (15).
Treatment with Proteasome Inhibitors-- Transfected COS-7 cells expressing mutant or wild-type insulin receptors were incubated in fetal calf serum-free DMEM with 0.3% Me2SO, 1% bovine serum albumin, and with 50 µM proteasome inhibitors, Z-Leu-Leu-Nva-H (MG-115), or Z-Leu-Leu-Leu-H (MG-132), or with 50 µM cysteine protease inhibitor E-64-d in CO2 incubator at 37 °C. After 2 h of incubation, the cells were washed twice with PBS and then dissolved in Laemmli sample buffer to be analyzed by Western blotting, as described above.
Microinjection Assay--
Cells were grown on glass coverslips
and rendered quiescent by starvation for 24 h in serum-free DMEM.
Antibodies in a buffer containing 5 mM NaPO4
and 100 mM KCl, pH 7.4, were microinjected into cells using
a glass capillary needle. Approximately 1 × 1014
liter of the buffer was introduced into each cell. The injection included about 1 × 106 molecules of IgG.
Microinjected cell numbers were 250-300/coverslip (26).
Immunofluorescent staining of the injected cells as described below
indicated that about 75% of the cells were successfully microinjected.
Immunocytochemical Staining-- Twelve hours after microinjection, the cells were fixed with 4% formaldehyde in PBS for 15 min at 22 °C. The fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min at 22 °C and blocked with a solution containing 2% bovine serum albumin and 0.1% NaN3 in PBS for 20 min at 22 °C. The cells were incubated with rat polyclonal anti-insulin receptor C-terminal (IRc) antibody in PBS containing 10 mM MgCl2 for 2 h at 22 °C. The cells were then incubated and stained with rhodamine-labeled donkey anti-rat IgG antibody and FITC-labeled donkey anti-rabbit IgG antibody for 1 h at 22 °C (26). After the coverslips were mounted, the cells were analyzed with a confocal laser microscope (Olympus, Japan).
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RESULTS |
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Accelerated Degradation of Mutant Insulin Proreceptor--
We
previously suggested that the accelerated degradation of both
Asp1179 and Leu1193 mutant insulin receptors
occurred in the stage of proreceptor (15). To examine whether
Asp1179 or Leu1193 mutation may cause the rapid
degradation of the proreceptors retained in the ER, we used the exon 13 deletion mutant (Ex13) of insulin receptor, which was accumulated in
the ER as the uncleaved proreceptor and could not undergo further
processing (22). We introduced Asp1179 or
Leu1193 mutation into the
Ex13 mutant insulin receptor
cDNAs, i.e.
Ex13+Asp1179 or
Ex13+Leu1193. After transfection of these cDNAs into
COS-7 cells, we examined the synthetic and processing time course of
these mutant receptors. These double mutant insulin receptors produce
only a single 190-kDa protein corresponding to the
Ex13 proreceptor
form (Fig. 1A). Insulin
binding to the COS-7 cells (1 × 106 cells)
transfected with wild-type, mock,
Ex13,
Ex13+Asp1179,
and
Ex13+Leu1193 cDNAs were 15 ± 0.9, 0.9 ± 0.3, 0.9, 0.8, and 0.9% of total, respectively. Therefore, both
double mutant receptors as well as the
Ex13 mutant receptor were not
transported to the cell surface. To investigate the degradation of
these mutant insulin proreceptors, steady-state labeling studies were
carried out in the transfected COS-7 cells. As shown in Fig. 1
(A and B), the labeled
Ex13 proreceptor slowly
degraded, and t1/2 for the
Ex13 proreceptor was
15 h (Fig. 1B). In contrast, both the
Ex13+Asp1179 and
Ex13+Leu1193 mutant
proreceptors disappeared rapidly with a half-life of 6 h, which
was 2.5 times faster than that for
Ex13 proreceptors. These results
indicated that both Asp1179 and Leu1193
mutations of the insulin receptor caused the accelerated degradation of
the unprocessed proreceptor accumulated in the ER.
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The Role of Proteasome in Degradation of the Mutant Insulin
Receptors Associated with the ER--
To investigate whether the
proteasome was involved in the mutant receptor degradation, we examined
the effect of proteasome inhibitors. The COS-7 cells expressing the
wild-type or the mutant insulin receptors were incubated with MG-115
(Z-Leu-Leu-Nva-H), MG-132 (Z-Leu-Leu-Leu-H), or E-64-d. Fig.
2 shows the two major bands of 190 and 95 kDa. The 190-kDa band corresponds to the proreceptor and 95-kDa band to
the mature -subunit. Treatment with E-64-d, which inhibited the
calpain and cathepsin protease activities, made no changes in the
intensities of the wild-type and mutant receptors. However, in the
Leu1193 and Asp1179 mutant cell lines,
treatment with both the proteasome inhibitors (MG-115 or MG-132)
increased the intensities of 190- and 95-kDa bands to 3- and 4.2-fold,
respectively. However, in the wild-type cell lines, there was no change
in the intensities of these bands by treatment with either proteasome
inhibitors or protease inhibitor (Fig. 2). These results suggest that
both Asp1179 and Leu1193 mutant insulin
proreceptors were degraded by the proteasome in the cytosol.
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Association of the Mutant Insulin Receptor with Heat Shock Protein 90-- It has been generally accepted that protein biosynthesis or degradation required the support of specific molecular chaperones (27, 28). We have reported previously the relationship between these mutant insulin receptors and Hsc70 or immunoglobulin heavy chain BiP (29). In the present study, we found that Hsp90 was also associated with both mutant receptors (Fig. 4, A and B), whereas calnexin and Hsp60 were not significantly associated with these mutants (data not shown). In the transfected COS-7 cells, wild-type or Asp1179 or Leu1193 mutant insulin receptors were immunoprecipitated with anti-Hsp90 antibody and analyzed by Western blotting with anti-insulin receptor C-terminal antibody. In Fig. 4A, the upper panel shows a 190-kDa band, which is the proreceptor co-immunoprecipitated with anti-Hsp90 antibody, and the lower panel indicates the insulin proreceptor of the same sample. In cells expressing the Asp1179 or Leu1193 mutant receptor, the amount of proreceptors associated with Hsp90 was 6- and 7-fold greater than that of the wild-type, respectively, when the amount of insulin receptor protein in each samples was adjusted.
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Microinjection of anti-Hsp90 Antibody to Transfected COS-7 Cells-- To investigate whether Hsp90 played an important role in the accelerated receptor degradation, we studied the effect of anti-Hsp90 antibody on the mutant receptor degradation. Insulin receptor was visualized by immunofluorescence microscopy utilizing FITC-labeled anti-rabbit IgG for anti-insulin receptor antibody (Fig. 5, left side), and injected cells were identified by rhodamine-labeled anti-mouse IgG antibody for anti-Hsp90 antibody (Fig. 5, A-D, right side), or rhodamine-labeled anti-rat IgG for anti-Hsc70 antibody (Fig. 5, E-H, right side). Microinjection of either anti-Hsp90 antibody or anti-Hsp70 antibody into COS-7 cells transfected with the wild-type insulin receptor cDNA (Fig. 5A) or mock (Fig. 5E) did not change the amount of insulin receptors. COS-7 cells, which were transfected with Asp1179 or Leu1193 mutant receptor cDNA, had a small amount of receptors compared with the cells transfected with wild-type cDNA (Fig. 5, B, C, F, and G; uninjected cells). However, microinjection of anti-Hsp90 antibody led to the clearly increased amount of the Asp1179 or Leu1193 mutant insulin receptors at 12 h after microinjection (Fig. 5, B and C; injected cells). On the other hand, microinjection of anti-Hsc70 (Fig. 5, F and G; injected cells) or anti-BiP antibody (data not shown) did not change the amount of the receptors.
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DISCUSSION |
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Most of the naturally occurring mutations in the -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
(16). Similarly, the mutant insulin receptor lacking the coding region
of exon 13 was not processed to the subunits and was also associated
with BiP (22, 29). Since this
Ex13 insulin receptor remained
unprocessed and was easily distinguished from normal matured insulin
receptors, we created double mutant insulin receptors that had both
Ex13 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
Ex13 insulin
receptor led to rapid receptor degradation (15), 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 (17, 18).
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 - and
-subunits takes place at the
Golgi apparatus.
The degradation by the proteasome usually requires polyubiquitination of the targeted proteins (18, 30-32). 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 (19, 33, 34).
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 (35, 36), pp60v-src (37, 38), Raf-1 (39), or casein kinase II (40). 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. (33) 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 -subunits (41) 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 (42),
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 (43, 44), it remains to be determined whether
they are also degraded in the same manner as described above. On the
other hand, in the
-subunit of the insulin receptor, only the
Glu460 mutation showed the accelerated receptor degradation
(14). 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.
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ACKNOWLEDGEMENT |
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We thank Prof. Yukio Ikehara (Department of Biochemistry, Fukuoka University School of Medicine, Japan) for helpful discussion and critical review of the manuscript.
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FOOTNOTES |
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* This work was supported in part by a research grant for intractable disease from the Ministry of Health and Welfare, a grant-in-aid from the Ministry of Education, Science, and Culture, and a grant for diabetes research from Otsuka Pharmaceutical Co. Ltd., Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 81-764-34-2281 (ext. 2500); Fax: 81-764-34-5025.
1 The 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.
2 The numbering of amino acids in this paper corresponds to the sequence of the receptor of Ebina et al. (2).
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REFERENCES |
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