1 Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
2 Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USA
*Author for correspondence (patricia_hinkle{at}urmc.rochester.edu)
Accepted July 9, 2001
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SUMMARY |
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Key words: Golgi apparatus, Growth hormone, Unfolded protein response
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INTRODUCTION |
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In addition to autosomal dominant GH deficiency, there are many other disorders in which misfolding of a protein leads to a deficiency or malfunction of the protein in question (Kuznetsov and Nigam, 1998; Perlmutter, 1999). When proteins misfold, they are often retained in the endoplasmic reticulum (ER) and targeted for degradation (Ellgaard et al., 1999). Cells can exhibit a variety of responses to the accumulation of misfolded protein in the ER. One such reaction, the unfolded protein response, involves the transcriptional activation of genes encoding a wide range of proteins necessary for protein folding and secretion (Chapman et al., 1998; Sidrauski et al., 1998; Mori, 2000; Travers et al., 2000). Other cellular strategies for handling accumulated misfolded protein have been demonstrated in association with specific misfolded proteins. For example, an accumulation of misfolded protein called an aggresome is formed when mutant cystic fibrosis transmembrane conductance regulator accumulates (Johnston et al., 1998). In some disorders, accumulation of misfolded protein in the ER may cause damage to the host cell (Teckman et al., 1996; Ito and Jameson, 1997).
These studies were designed to learn how cells respond to the expression of the misfolded 32-71-GH and whether intracellular accumulation of the
32-71-GH protein has consequences that might damage a somatotroph over time and contribute to the dominant negative phenotype. Here we show that the misfolded GH, when expressed in COS cells, is retained in the ER. Furthermore, we provide direct experimental evidence that the expression of misfolded GH causes fragmentation of the Golgi apparatus and interferes with the trafficking of other, nonmutant proteins, and that these effects may be due to failure of
32-71-GH to induce an adequate unfolded protein response. These findings provide a possible mechanism for cellular toxicity in some diseases of protein misfolding, demonstrate the heterogeneity of cellular responses to misfolded proteins and suggest possible therapeutic strategies for prevention of cellular damage in clinical disorders associated with ER protein accumulation.
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MATERIALS AND METHODS |
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For northern blots, total RNA was isolated by RNeasy (Qiagen) and 5 µg RNA were loaded per lane. The probe for BiP was a 1.5 kb EcoR1-PstI fragment from p3C5, a plasmid containing the cDNA for hamster BiP (from Amy S. Lee, University of Southern California, Los Angeles, CA), and labeled by random priming. For western blots, transfected cells were lysed in Laemmli sample buffer and proteins were resolved by SDS-PAGE on 12% gels. Proteins were transferred to nitrocellulose and blots incubated with antibody to GH or ß-COP (1:1000). Bands were identified using ECL (NEN Life Science Products, Boston, MA), for ß-COP, or [125I]protein A (NEN) for GH. Specific binding of 5 nM [3H]MeTRH was measured at 37°C as described (Yu and Hinkle, 1998).
For Hoechst staining, methanol:acetone-fixed cells were incubated for 5 minutes in a 1.5 µg/ml aqueous solution of Hoechst 33432 (Molecular Probes). In some experiments, Hoechst staining was done after immunocytochemical staining for GH. Staining was observed using a UV-1A filter set from Chroma Technology (Brattleboro, VT).
A plasmid encoding secreted alkaline phosphatase (SEAP) cloned in pcDNA3 was obtained from Sven-Ulrik Gorr, University of Louisville School of Dentistry, Louisville, KY. Cells were transfected with 2.5 µg SEAP and 2.5 µg either wt-GH or 32-71-GH. Medium was collected 24 or 48 hours after transfection and secreted enzyme activity was measured using the Phospha-Light chemiluminescent assay from Tropix (Foster City, CA). The amount of SEAP activity obtained when cells were cotransfected with wt-GH was set at 100%.
The measurements of prolactin synthesis in cells cotransfected with wt-GH or 32-71-GH were performed by [35S]amino acid incorporation for 10 minutes followed by immunoprecipitation from the cell lysate and gel electrophoresis as described (Lee et al., 2000).
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RESULTS |
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Effect of 32-71-GH on cytoplasmic microtubules
Golgi stacks in COS cells are normally juxtaposed to the centrosome, the major organizing center for microtubules in the cytoplasm (Thyberg and Moskalewski, 1999), and microtubule depolymerization leads to redistribution of the Golgi from the centrosomal location to peripheral sites of protein exit from the ER (Cole et al., 1996). Therefore, we asked if the Golgi fragmentation induced by expression of 32-71-GH was associated with changes in cytoplasmic microtubular arrangement. Staining for
-tubulin revealed no microtubule depolymerization in cells expressing either wt-GH or
32-71-GH. Most cells expressing wt-GH had well-defined microtubule-organizing centers typical of normal interphase cells. However, most cells expressing
32-71-GH did not contain distinct microtubule-organizing centers (Fig. 4). Visual counting of wt-GH-positive cells and
32-71-GH-positive cells (80 cells from two separate experiments for each GH construct) revealed that 76% of cells expressing wt-GH had obvious microtubule-organizing centers, whereas this microtubular pattern was seen in only 8% of cells expressing
32-71-GH.
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32-71-GH weakly induces the unfolded protein response
To determine whether 32-71-GH induces the unfolded protein response, we used northern analysis to quantify the amount of mRNA for BiP, also called GRP78, in cells transfected with empty vector or plasmids encoding wt-GH or
32-71-GH. BiP was induced only weakly and to the same extent (twofold) by wild-type or misfolded GH. BiP was induced strongly (11-fold) by treatment of COS cells with dithiothreitol (DTT), a strong reducing agent known to induce the unfolded protein response (Molinari and Helenius, 1999) (Fig. 7). Tunicamycin, which interferes with glycoprotein synthesis and is also used to induce the unfolded protein response, caused fourfold and sevenfold increases in BiP mRNA when used at 1 µg/ml and 10 µg/ml, respectively. Expression of a mutant insulin that does not fold properly increased BiP mRNA fivefold (Fig. 7). These experiments show that
32-71-GH does not markedly induce the unfolded protein response, which would be expected to help protect the cells against toxic effects resulting from unfolded proteins.
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Effect of 32-71-GH on nuclear chromatin pattern
Golgi fragmentation occurs during apoptosis (Sesso et al., 1999). We tested whether 32-71-GH induces condensation and fragmentation of nuclear chromatin to investigate the possibility that the
32-71-GH-induced Golgi fragmentation was secondary to an overall increase in cell death by apoptosis. In one experiment, cells were transfected with either wt-GH,
32-71-GH, empty pcDNA3 vector or, as a positive control, with epitope-tagged bax, a strong inducer of apoptosis. After 24 hours, cells were costained for either GH (wt-GH and
32-71-GH transfections) or the FLAG epitope (bax-FLAG transfections) and with Hoechst dye, which identifies nuclear chromatin, and scored for condensed or fragmented nuclear chromatin, a consistent characteristic of apoptotic cells. Apoptotic nuclei were observed in 177/200 (88.5%) of cells expressing bax, but in only 35/200 (17.5%) and 39/200 (19.5%) of cells expressing wt- or
32-71-GH, respectively. No GH or FLAG immunoreactivity was seen in cells transfected with the empty plasmid. These results suggest that expression of
32-71-GH does not increase apoptosis as compared to cells expressing wt-GH and that the Golgi fragmentation we observed is probably not due to the general disassembly of cellular organelles reported in cells undergoing apoptosis (Kohler et al., 1990).
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DISCUSSION |
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We examined the expression of 32-71-GH in COS cells, and found that this misfolded protein, like many others, accumulates in the ER. We further found that expression of
32-71-GH leads to fragmentation of the Golgi apparatus and aberrant trafficking of other proteins destined for the plasma membrane or for secretion. Such changes seem likely to lead to cellular toxicity and may provide an explanation for why some proteins that cannot fold correctly exert dominant negative effects.
In previous studies, we found that 32-71-GH did not accumulate in several neuroendocrine cell lines over a 24 hour period (Lee et al., 2000). Our finding that cotransfected
32-71-GH accumulated in COS cells and caused altered trafficking of prolactin may reflect differences in protein expression and secretory pathways between cell types.
32-71-GH may resemble
1-ATZ in that it becomes more toxic in situations where it accumulates. The neuroendocrine cells lines, however, may not be good models for the normal somatotroph through all its stages of development and maturation. If expression of
32-71-GH leads to degradation of Golgi structure and function, similar to what we have observed, at any stage of maturation, this disruption would be expected over time to damage or destroy pituitary somatotrophs. This presents a plausible explanation for the dominant phenotype seen in isolated GH deficiency type II. This hypothesis is supported by recent studies in transgenic mice in which expression of
32-71-GH caused selective disappearance of somatotrophs by 3 weeks of age (I. C. Robinson, personal communication). Histological studies of pituitary glands from people heterozygous for the
32-71-GH mutation have not been reported.
Transport of proteins from the ER occurs in COPII vesicles that bud from selected sites, and some proteins that cannot achieve their normal configuration accumulate at these sites (Raposo et al., 1995). Aridor et al. used transmission electron microscopy and morphometric analysis to show that expression of a temperature-sensitive mutant of vesicular stomatitis glycoprotein (VSV-G) or 1-ATZ reduced the number of vesicles budding from the ER (Aridor et al., 1999). These experiments suggest that correctly folded and assembled cargo proteins are involved in the formation of their own COPII-coated ER-to-Golgi transport vesicles. Studies examining formation of COPII vesicles in vitro supported this conclusion. The disruption of protein transport and Golgi morphology caused by
32-71-GH in our studies could result from a similar failure of COPII vesicles to bud. Additional studies are needed to pinpoint the mechanism underlying abnormal protein trafficking in cells expressing
32-71-GH. In light of the results of Aridor et al. (Aridor et al., 1999), incorrectly folded proteins could interfere with transport of other, normal proteins out of the ER. Our findings that
32-71-GH interferes with the flow of prolactin, TRH receptors and secretory alkaline phosphatase confirm this prediction. It is important to note that not all incorrectly folded proteins disrupt protein transport and Golgi morphology. In these studies, we found that an insulin mutant accumulates in the ER but does not alter Golgi staining. In another example, Hobman et al. examined accumulation of Rubella virus E1 glycoprotein in the ER (Hobman et al., 1998); E1 is only transported further along the secretory pathway if it is complexed with E2. They found that E1 accumulates at specific sites of exit from ER but does not affect the distribution of COPII staining.
Golgi fragmentation has been observed previously as a late step in cells undergoing apoptosis (Watanabe et al., 2000). We considered the possibility that the Golgi fragmentation we observed was secondary to apoptosis induced by 32-71-GH, but we did not detect apoptotic features in cells expressing the misfolded hormone.
Another well-known condition associated with Golgi fragmentation is microtubule depolymerization (Cole et al., 1996). When microtubule polymers are disrupted, Golgi membrane components redistribute to peripheral sites of protein exit from the ER; Golgi function is preserved under these conditions despite the structural changes. The patterns of -tubulin staining we observed in cells expressing
32-71-GH, however, are not consistent with microtubular depolymerization. Rather, microtubule-organizing centers were disrupted. Similar disordering of cytoplasmic microtubules has been seen in cells infected with vaccinia virus (Ploubidou et al., 2000) and in cells treated with a DNA polymerase inhibitor (Tanaka et al., 1998). The effects of
32-71-GH and other misfolded proteins on microtubular dynamics bears further study. Rowe et al. reported disassembly of the Golgi apparatus in neurosecretion-defective PC12 clones and NRK fibroblasts transfected with cDNA encoding syntaxin 1A, a t-SNARE involved in synaptic exocytosis (Rowe et al., 1999). This Golgi disassembly was prevented by cotransfection of rbSec1, a protein that interacts with syntaxin 1A in neurosecretory exocytosis. It is unlikely that the Golgi fragmentation in cells expressing
32-71-GH was due to absence of a protein like rbSec1, however, as Golgi disassembly did not occur in cells expressing either wt-GH or misfolded insulin.
Golgi fragmentation has also been reported in cells treated with 1-butanol, an inhibitor of phosphatidylinositol(4,5)bisphosphate formation (Siddhanta et al., 2000). It will be of interest to determine whether phosphatidylinositol(4,5)bisphosphate concentrations are reduced in cells expressing 32-71-GH.
Not all proteins that aggregate in the ER are toxic. For example, the Mody mouse has an autosomal dominant mutation that results in a Tyr for Cys substitution in insulin 2. This mutation prevents proper folding and processing of wild-type insulins 1 and 2, so that they aggregate in the ER, but the pancreatic ß cells are not destroyed, and insulin mRNA is transcribed normally (Wang et al., 1999). Mice with this mutant overexpress BiP, a molecular chaperone in the ER (Wang et al., 1999). By contrast, 32-71-GH causes little or no induction of BiP, and in this respect resembles the
1-ATZ variant, which also accumulates in the ER without inducing BiP mRNA (Cresteil et al., 1990; Graham et al., 1990). Although BiP binds to many unfolded proteins, Graham et al. were unable to detect BiP binding to
1-ATZ (Graham et al., 1990), and we have been unable to detect BiP binding to
32-71-GH (T.K.G. and P.M.H., unpublished). The unfolded protein response, including increased transcription of BiP mRNA, is induced by activation of the transmembrane kinase Ire1p, and binding of BiP to the lumenal portion of Ire1p is thought to keep the kinase from dimerizing and activating itself (Bertolotti et al., 2000). If this activation mechanism is correct, proteins that fail to bind BiP will not induce the unfolded protein response because they fail to compete with BiP binding to Ire1p. Over 300 genes are induced in the unfolded protein response in yeast, and many of these gene products have functions in the secretory pathway, including translocation into the ER, protein folding and modification, protein degradation through the proteasome pathway, and transport from the ER to the Golgi complex and from the Golgi complex to lysosomes or the cell surface (Travers et al., 2000). Thus, the unfolded protein response is a useful protective mechanism against damage resulting from unfolded proteins. Proteins such as
32-71-GH and
1-ATZ that induce the unfolded protein response poorly are more likely to cause cellular toxicity. This is supported by our finding that strategies to induce the unfolded protein response decreased Golgi apparatus fragmentation, and partially rescued normal TRH receptor trafficking in cells expressing mutant GH. It is notable that induction of the unfolded protein response with tunicamycin, as well as by expression of a misfolded insulin mutant, gave similar results. It will be of interest to examine whether the failure to induce the unfolded protein response contributes to the mechanism of cytotoxicity seen in other protein misfolding diseases. It will also be interesting to test the notion that therapeutic induction of the unfolded protein response might alleviate the cellular damage caused by the accumulation of the misfolded protein.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Aridor, M., Bannykh, S. I., Rowe, T. and Balch, W. E. (1999). Cargo can modulate COPII vesicle formation from the endoplasmic reticulum. J. Biol. Chem. 274, 4389-4399.
Ashworth, R., Yu, R., Nelson, E. J., Dermer, S., Gershengorn, M. C. and Hinkle, P. M. (1995). Visualization of the thyrotropin-releasing hormone receptor and its ligand during endocytosis and recycling. Proc. Natl. Acad. Sci. USA 92, 512-516.[Abstract]
Berger, J., Hauber, J., Hauber, R., Geiger, R. and Cullen, B. R. (1988). Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66, 1-10.[Medline]
Bertolotti, A., Zhang, Y., Hendershot, L. M., Harding, H. P. and Ron, D. (2000). Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat. Cell Biol. 2, 326-332.[Medline]
Chapman, R., Sidrauski, C. and Walter, P. (1998). Intracellular signaling from the endoplasmic reticulum to the nucleus. Annu. Rev. Cell Dev. Biol. 14, 459-485.[Medline]
Cogan, J. D., Phillips, J. A., III, Schenkman, S. S., Milner, R. D. and Sakati, N. (1994). Familial growth hormone deficiency: a model of dominant and recessive mutations affecting a monomeric protein. J. Clin. Endocrinol. Metab. 79, 1261-1265.[Abstract]
Cogan, J. D., Ramel, B., Lehto, M., Phillips, J., III, Prince, M., Blizzard, R. M., de Ravel, T. J., Brammert, M. and Groop, L. (1995). A recurring dominant negative mutation causes autosomal dominant growth hormone deficiency-a clinical research center study. J. Clin. Endocrinol. Metab. 80, 3591-3595.[Abstract]
Cole, N. B., Sciaky, N., Marotta, A., Song, J. and Lippincott-Schwartz, J. (1996). Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites. Mol. Biol. Cell 7, 631-650.[Abstract]
Cresteil, D., Ciccarelli, E., Soni, T., Alonso, M. A., Jacobs, P., Bollen, A. and Alvarez, F. (1990). BiP expression is not increased by the accumulation of PiZ alpha 1-antitrypsin in the endoplasmic reticulum. FEBS Lett. 267, 277-280.[Medline]
de Vos, A. M., Ultsch, M. and Kossiakoff, A. A. (1992). Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 255, 306-312.[Medline]
Ellgaard, L., Molinari, M. and Helenius, A. (1999). Setting the standards: quality control in the secretory pathway. Science 286, 1882-1888.
Gao, Y. S., Alvarez, C., Nelson, D. S. and Sztul, E. (1998). Molecular cloning, characterization, and dynamics of rat formiminotransferase cyclodeaminase, a Golgi-associated 58-kDa protein. J. Biol. Chem. 273, 33825-33834.
Graham, K. S., Le, A. and Sifers, R. N. (1990). Accumulation of the insoluble PiZ variant of human alpha 1-antitrypsin within the hepatic endoplasmic reticulum does not elevate the steady-state level of grp78/BiP. J. Biol. Chem. 265, 20463-20468.
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. and Pease, L. R. (1989). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51-59.[Medline]
Hobman, T. C., Zhao, B., Chan, H. and Farquhar, M. G. (1998). Immunoisolation and characterization of a subdomain of the endoplasmic reticulum that concentrates proteins involved in COPII vesicle biogenesis. Mol. Biol. Cell 9, 1265-1278.
Ito, M. and Jameson, J. L. (1997). Molecular basis of autosomal dominant neurohypophyseal diabetes insipidus. Cellular toxicity caused by the accumulation of mutant vasopressin precursors within the endoplasmic reticulum. J. Clin. Invest. 99, 1897-1905.
Johnston, J. A., Ward, C. L. and Kopito, R. R. (1998). Aggresomes: a cellular response to misfolded proteins. J. Cell Biol. 143, 1883-1898.
Kaushal, S. and Khorana, H. G. (1994). Structure and function in rhodopsin. 7. Point mutations associated with autosomal dominant retinitis pigmentosa. Biochemistry 33, 6121-6128.[Medline]
Kim, P. S. and Arvan, P. (1998). Endocrinopathies in the family of endoplasmic reticulum (ER) storage diseases: disorders of protein trafficking and the role of ER molecular chaperones. Endocr. Rev. 19, 173-202.
Kohler, H. R., Dhein, J., Alberti, G. and Krammer, P. H. (1990). Ultrastructural analysis of apoptosis induced by the monoclonal antibody anti-APO-1 on a lymphoblastoid B cell line. Ultrastruct. Pathol. 14, 513-518.[Medline]
Kuznetsov, G. and Nigam, S. K. (1998). Folding of secretory and membrane proteins. New Engl. J. Med. 339, 1688-1695.
Lee, M. S., Wajnrajch, M. P., Kim, S. S., Plotnick, L. P., Wang, J., Gertner, J. M., Leibel, R. L. and Dannies, P. S. (2000). Autosomal dominant growth hormone (GH) deficiency type II: the Del32-71-GH deletion mutant suppresses secretion of wild-type GH. Endocrinology 141, 883-890.
Lowe, S. L., Peter, F., Subramaniam, V. N., Wong, S. H. and Hong, W. (1997). A SNARE involved in protein transport through the Golgi apparatus. Nature 389, 881-884.[Medline]
Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M. and Tsien, R. Y. (1997). Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882-887.[Medline]
Molinari, M. and Helenius, A. (1999). Glycoproteins form mixed disulphides with oxidoreductases during folding in living cells. Nature 402, 90-93.[Medline]
Mori, K. (2000). Tripartite management of unfolded proteins in the endoplasmic reticulum. Cell 101, 451-454.[Medline]
Oprins, A., Duden, R., Kreis, T. E., Geuze, H. J. and Slot, J. W. (1993). Beta-COP localizes mainly to the cis-Golgi side in exocrine pancreas. J. Cell Biol. 121, 49-59.[Abstract]
Perlmutter, D. H. (1999). Misfolded proteins in the endoplasmic reticulum. Lab. Invest. 79, 623-638.[Medline]
Phillips, J. A., III and Cogan, J. D. (1994). Genetic basis of endocrine disease. 6. Molecular basis of familial human growth hormone deficiency. J. Clin. Endocrinol. Metab. 78, 11-16.[Medline]
Ploubidou, A., Moreau, V., Ashman, K., Reckmann, I., Gonzalez, C. and Way, M. (2000). Vaccinia virus infection disrupts microtubule organization and centrosome function. EMBO J. 19, 3932-3944.
Raposo, G., van Santen, H. M., Leijendekker, R., Geuze, H. J. and Ploegh, H. L. (1995). Misfolded major histocompatibility complex class I molecules accumulate in an expanded ER-Golgi intermediate compartment. J. Cell Biol. 131, 1403-1419.[Abstract]
Rowe, J., Corradi, N., Malosio, M. L., Taverna, E., Halban, P., Meldolesi, J. and Rosa, P. (1999). Blockade of membrane transport and disassembly of the Golgi complex by expression of syntaxin 1A in neurosecretion-incompetent cells: prevention by rbSEC1. J. Cell Sci. 112, 1865-1877.
Sesso, A., Fujiwara, D. T., Jaeger, M., Jaeger, R., Li, T. C., Monteiro, M. M., Correa, H., Ferreira, M. A., Schumacher, R. I., Belisario, J. et al. (1999). Structural elements common to mitosis and apoptosis. Tissue Cell 31, 357-371.[Medline]
Siddhanta, A., Backer, J. M. and Shields, D. (2000). Inhibition of phosphatidic acid synthesis alters the structure of the Golgi apparatus and inhibits secretion in endocrine cells. J. Biol. Chem. 275, 12023-12031.
Sidrauski, C., Chapman, R. and Walter, P. (1998). The unfolded protein response: an intracellular signalling pathway with many surprising features. Trends Cell Biol. 8, 245-249.[Medline]
Sung, C. H., Davenport, C. M. and Nathans, J. (1993). Rhodopsin mutations responsible for autosomal dominant retinitis pigmentosa. Clustering of functional classes along the polypeptide chain. J. Biol. Chem. 268, 26645-26649.
Tanaka, H., Takenaka, H., Yamao, F. and Yagura, T. (1998). Aphidicolin induces alterations in Golgi complex and disorganization of microtubules of HeLa cells upon long-term administration. J. Cell Physiol. 176, 602-611.[Medline]
Teckman, J. H., Qu, D. and Perlmutter, D. H. (1996). Molecular pathogenesis of liver disease in alpha1-antitrypsin deficiency. Hepatology 24, 1504-1516.[Medline]
Thyberg, J. and Moskalewski, S. (1999). Role of microtubules in the organization of the Golgi complex. Exp. Cell Res. 246, 263-279.[Medline]
Travers, K. J., Patil, C. K., Wodicka, L., Lockhart, D. J., Weissman, J. S. and Walter, P. (2000). Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101, 249-258.[Medline]
Wang, J., Takeuchi, T., Tanaka, S., Kubo, S. K., Kayo, T., Lu, D., Takata, K., Koizumi, A. and Izumi, T. (1999). A mutation in the insulin 2 gene induces diabetes with severe pancreatic beta-cell dysfunction in the Mody mouse. J. Clin. Invest. 103, 27-37.
Watanabe, J., Amizuka, N., Noda, T. and Ozawa, H. (2000). Cytochemical and ultrastructural examination of apoptotic odontoclasts induced by bisphosphonate administration. Cell Tissue Res. 301, 375-387.[Medline]
Wu, Y., Whitman, I., Molmenti, E., Moore, K., Hippenmeyer, P. and Perlmutter, D. H. (1994). A lag in intracellular degradation of mutant alpha 1-antitrypsin correlates with the liver disease phenotype in homozygous PiZZ alpha 1-antitrypsin deficiency. Proc. Natl. Acad. Sci. USA 91, 9014-9018.[Abstract]
Yu, R. and Hinkle, P. M. (1998). Signal transduction, desensitization, and recovery of responses to thyrotropin-releasing hormone after inhibition of receptor internalization. Mol. Endocrinol. 12, 737-749.