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Limited Degradation of Retinoid X Receptor by Calpain
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
225, 946–951 (1996)
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Limited Degradation of Retinoid X Receptor by Calpain Rie Matsushima-Nishiwaki, Yoshihiro Shidoji, Shinji Nishiwaki, Hisataka Moriwaki, and Yasutoshi Muto1 First Department of Internal Medicine, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu city, Gifu 500, Japan Received July 15, 1996 Recently, we have found two major physiological forms of retinoid X receptor a (RXRa): the mature 54 kDa RXRa and the truncated 44 kDa RXRa lacking a portion of N-terminal A/B domain in human and rodent livers. In this communication, we show that m-calpain was active to digest 54 kDa RXRa in the human hepatoma-derived cell line, HuH7, nuclei to 44 kDa fragment through 47 kDa intermediate in vitro. Although both proteolytic fragments were revealed by anti-RXRa antibody against its E-domain, neither fragment reacted with anti-RXRa antibody specific for A/B domain. The profile of the calpaininduced proteolytic fragmentation of RXRa was almost identical to that of endogenous RXRa in nonmalignant human and normal mouse liver nuclei. This is the first demonstration that RXRa is a substrate for mcalpain, strongly suggesting that the enzyme might also be involved in post-translational modification of the receptor in hepatocytes. q 1996 Academic Press, Inc.
Retinoid X receptors (RXRs) belong to nuclear receptor superfamily, bind 9-cis retinoic acid as an endogenous ligand, and regulate several gene expressions (1-3). RXR forms homodimer and heterodimers with retinoic acid receptors, vitamin D receptor, thyroid hormone receptors, peroxisome proliferator-activated receptors or other orphan receptors (4) and is now recognized to play a central role in the biologic function of the nuclear receptor superfamily. These dimers bind their response elements and subsequently activate or inhibit the expression of their target genes (5). There are three RXR subtypes (a, b and g) and these subtypes show the different organ expression patterns (5). RXRa is the most abundant in the liver (6) and is also highly expressed in human hepatocellular carcinoma (HCC)-derived HuH7 cells (7, 8). RXRs are characterized by a modular domain structure and they contain the A through E regions (9). The non-conserved N-terminal A/B domain has an autonomous transcriptional activation function (AF-1), a fairly conserved C domain shows DNA binding activity, and beyond a hinge domain D, the variable C-terminal E domain gives ligand binding pocket (10) and dimerization activities with ligand dependent activation function (AF-2) (11, 12). Recently, we have found the two major physiological forms of the nuclear RXRa; the mature 54 kDa RXRa and the truncated 44 kDa RXRa lacking a portion of amino-terminal A/B domain in the human and mouse livers and, furthermore, we have shown that the intranuclear metabolism from 54 kDa to 44 kDa RXR was impaired in HCC compared with its surrounding non-malignant tissues (13). Even though we are still unaware of the functional aspect in the proteolytic fragmentation, it is intriguing for us to study the enzymatic regulation of molecular conversion from 54 kDa to 44 kDa RXRa in order to scrutinize irreversible posttranslational transformation of nuclear transcription factors in general. 1 To whom correspondence should be addressed. Fax: /81-58-262-8484. Abbreviations used: RXR, retinoid X receptor; HCC, human hepatocellular carcinoma; EDTA, ethylene diamine tetraacetic acid; SDS, sodium dodecyl sulfate; ECL, enhanced chemiluminescence; RXRE, retinoid X receptor response element.
946 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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In the literatures, studies on proteolytic cleavage of nuclear transcription factors are scanty, but, Watt and Molloy (14) have found that a number of sequence-specific transcription factors such as USF, AP-1, Pit-1, Oct-1, CP1a, c-Myc, CREB, AP-2 and AP-3, but not Sp-1 were limitedly cleaved by nonlysosomal calcium-dependent cysteine protease, m-calpain. The calpain is distributed in ubiquitous tissues and cells, but its substrates are relatively limited (15). Since these include nuclear hormone-receptors such as progesterone receptor or estrogen receptor (16) as well as other transcription factors (14, 17), we were very much interested to know whether m-calpain is involved in production of 44 kDa RXRa or not. In this communication, we show that the 54 kDa RXRa was in vitro digested at A/B domain into 44 kDa RXRa by m-calpain through 47 kDa intermediate. MATERIALS AND METHODS Preparation of nuclear extracts. Nuclear extracts were prepared from HuH7 cells with or without protease inhibitors (leupeptin, pepstatin A, chymostatin and a-1 antitrypsin) and also from mouse primary cultured hepatocytes, according to the method of Rochette-Egly et al (18) with minor modifications. Mouse hepatocytes were isolated from male CD1 mice livers digested with collagenase (19) and cultured on a plastic flask. Determination of protein concentrations was conducted according to the method of Bradford (20). Digestion of the nuclear extracts with m-calpain. m-Calpain was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Digestion was conducted according to the method of Hirai et al (17). Briefly, the nuclear extracts of HuH7 cells were mixed on ice with a calpain solution (5 ml), and the reaction was started by adding 5 ml of the reaction mixture (0.4 M Tris-HCl, pH 7.8, 12 mM CaCl2 , and 2 mM dithiothreithol) at 307C. In some experiments, CaCl2 in reaction mixture was replaced with 12 mM ethylene diamine tetraacetic acid (EDTA). After incubation for 10 min., 4% sodium dodecyl sulfate (SDS) sample buffer (20 ml) was added and boiled for 5 min to stop the reaction. Western blot analysis. The boiled samples were electrophoresed on a 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane. The immunodetection was performed with polyclonal anti-RXRa(E) or D-20. Polyclonal anti-RXRa(E) was raised against E domain of the recombinant human RXRa as described elsewhere (13), and D-20, the polyclonal antibody for A/B domain of human RXRa was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). And the membrane was submitted to the enhanced chemiluminescence (ECL) detection, according to the protocol provided by Amersham, Buckinghamshire, UK. After exposure of the blotting membrane to an X-ray film, the film was scanned by JX-330M scanner (Sharp, Osaka, Japan) and densitometric analyses were conducted by using the NIH Image version 1.59 software. PEST sequence analysis. PEST sequence analysis was performed using PEST sequence searching program provided through Internet (http://www.biu.icnet.uk/projects/pest/) by Dr. Rechsteiner, University of Utah.
RESULTS AND DISCUSSION
As shown in Figure 1A, the major form of RXRa in human HCC-derived HuH7 cells was 54 kDa, predicted size from cDNA sequence of RXRa and minor bands of 47 kDa and 44 kDa were also detected. In a sharp contrast, the major form of RXRa in normal mouse primary cultured hepatocytes was found as 44 kDa with a faint band of 54 kDa RXRa, even-though a cocktail of protease inhibitors were included during the preparation of the nuclear extracts. The 44 kDa RXRa of mouse hepatocytes as well as 47 kDa and 44 kDa RXRa of HuH7 cells did not react with D-20, anti-A/B domain of RXRa, whereas the antibody detected 54 kDa species on the same filter (Figure 1B). This would suggest that the both 47 kDa and 44 kDa RXRa lack a portion of A/B domain and are the intranuclear proteolytic fragments of the mature 54 kDa RXRa. The previous reports on proteases responsible for proteolytic cleavage of nuclear receptors were limited, but progesterone receptor and estrogen receptor are substrates for m-calpain which associates subcellular organelles including the nuclear fraction (16, 21, 22). Although, it is an isozyme that requires a calcium concentration of ú0.2 mM for full activity in vitro (23), it was found to reach half maximal activity as 3 mM Ca2/ in isolated rat liver nuclei (24). Therefore, we prepared the nuclear extracts of HuH7 cells in the absence of protease inhibitors and then added exogenous m-calpain to the extracts. Compared with lane 1 in Figure 1A, a slight increase in 47 kDa and 44 kDa RXRa was already found in the nuclear extracts 947
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FIG. 1. Western blotting analyses of the intranuclear molecular species of RXRa. The nuclear extracts prepared with protease inhibitors from human hepatocellular carcinoma derived HuH7 cells (lanes 1) and from mouse primary cultured hepatocytes (lanes 2) were submitted to immunoblotting using anti-hRXRa(E) (panel A) and D-20, antiA/B domain of hRXRa (panel B). Arrow indicates the mobility of 54 kDa, arrow with dotted line indicates that of 47 kDa, and arrowhead indicates that of 44 kDa.
incubated with 3 mM Ca2/ and without protease inhibitors (Figure 2A, lane 1), suggesting that the nuclear extracts may contain endogenous Ca2/-activated protease activity. Exogenously added m-calpain attacked 54 kDa RXRa as well as 47 kDa RXRa, but 44 kDa RXRa was quite stable even in the presence of 100 mg/ml calpain (Figure 2A, lane 5). The amount of 54 kDa RXRa decreased in accordance with the increase of 47 kDa RXRa in the concentration dependent manner of m-calpain added. m-Calpain cleaved almost all of the 54 and 47 kDa RXRa into 44 kDa RXRa at the concentration of 100 mg/ml. In the absence of the Ca2/, the proportion of the 54, 47 and 44 kDa RXRa in the nuclear extract of HuH7 cells was not
FIG. 2. Concentration dependence of m-calpain-induced proteolytic profiles of RXRa in the nuclear extracts. (A) The nuclear extracts from HuH7 cells, prepared without protease inhibitors, were incubated with the indicated concentrations of m-calpain in the presence or absence of Ca2/. After 10 min incubation at 307C, the digests were separated in a 10% SDS-polyacrylamide gel and the RXRas were detected by immunoblotting with anti-hRXRa(E). (B) Percent distribution of each RXRa species to total RXRa in each lane of (A) was calculated after densitmetric analysis. 54 kDa (l), 47 kDa (n), 44 kDa (s). 948
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FIG. 3. Lack of reactivity with anti-A/B domain antibody in the calpain-induced fragments of RXRa. RXRa was treated with m-calpain in the presence (lanes 3) or absence (lanes 2) of Ca2/ and submitted to immunoblotting using anti-hRXRa(E) (panel A) or D-20, anti-A/B domain of RXRa (panel B).
changed even at 100 mg/ml m-calpain (Figure 2A). Thus, the digestion from 54 kDa RXRa to 47 kDa and 44 kDa fragment was absolutely Ca2/-dependent, excluding the possibility that other protease activity contaminated in m-calpain preparation may digest 54 kDa RXRa. The densitometric analysis (Figure 2B) clearly shows that 47 kDa RXRa was an intermediate form produced on the way of the digestion from 54 kDa RXRa to 44 kDa. As mentioned above, the physiologically truncated forms of 47 and 44 kDa RXRa lacked a portion of A/B domain. The molecular sizes of m-calpain-induced products were almost identical to those of the physiological fragments of RXRa, however, we had to determine whether the calpain-products were lacking in A/B domain from the 54 kDa mature RXRa of the nuclear extract of HuH7 cells. As shown in Figure 3, the 44 kDa RXRa which reacted with the anti-RXRa(E) antibody failed to react with D-20 antibody, so did the 47 kDa RXRa under the condition where the antibody detected 54 kDa RXRa. Therefore, the 44 and 47 kDa RXRa are supposed to be digested from 54 kDa RXRa by m-calpain at A/B domain, and are relevant to the physiologically truncated forms of RXRa. It has been proposed that the amino acid sequences of most short-lived proteins include one or more regions rich in proline (P), glutamic acid (E), aspartic acid, serine (S) and threonine (T). These regions, known as ‘PEST’ regions, may be recognized directly or indirectly by calpain, which hydrolyzes a peptide bond at the vicinity of PEST region (25). The sequence of the PEST region of RXRs were searched by using the PEST-FIND program provided through Internet by Dr. Rechsteiner, University of Utah, UT, USA (25) (Figure 4). Two potential PEST sequences were found at amino acids 75-108 mapping at carboxy terminus half of A/B domain and 215-234 mapping at carboxy terminus half of hinge D domain of human, mouse and rat RXRa. One can reasonably speculate that m-calpain may digest at the vicinity of the first potential PEST region resided on A/B domain of 54 kDa RXRa to produce 47 kDa RXRa, leaving DNA-binding C domain intact. The sequence of amino acids 75-108 was highly conserved between RXR subtypes. The same region in A/B domain in RXRb was also the potential PEST sequence, and in RXRg, it was poor but candidate PEST sequence. 949
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FIG. 4. Diagrammatic representation of PEST regions in the amino acid sequences of RXRas, RXRbs, and RXRg. The possible PEST regions (0 õ PEST-FIND score) are indicated by closed boxes, and the poor candidate PEST regions (0 § PEST-FIND score) are indicated by open boxes. PEST regions were searched by PEST-FIND program as described in Materials and Methods.
To produce 44 kDa RXRa from 47 kDa RXRa, a candidate PEST sequence of amino acids 440-459 mapping at amino terminus of human RXRa is the most probable site for digestion. Even though the region is poor candidate PEST sequence, the most carboxy terminal region called tc is three dimensionally extended to outside from the core structure of the RXRa, thus it must be easily accessible to the protease. The most carboxy terminal region of the the RXRs is also highly conserved, and the regions in RXRb and g were also poor but candidate PEST sequence. This carboxy terminus region is very important position for the ligand dependent transcription, named as AF-2 (4). Another ligand independent transcription mediated region, AF-1 is located at A/B domain of RXRs (4). Taking account of these functional modular domains of RXR, the present results suggest that RXRa may lose AF-1 at the initial step of the proteolysis and thereafter lose AF-2 at the second step from 47 kDa to 44 kDa digestion by m-calpain, and these proteolyses may be happened to not only RXRa but also to b and g. According to the report that the recombinant RXRDAB, lacking in A/B domain, hetero- or homo-dimerized and binded to synthetic direct repeats (26), 47 kDa and 44 kDa RXR found in calpain-digests may well bind to natural response elements. In conclusion, m-calpain cleaved out an N-terminal portion of A/B domain from the mature RXRa to produce 47 kDa and 44 kDa fragments. This unique irreversible two-step transformation of RXRa might be important for the signal transduction of retinoids. The exact digestion sites and the role of the 47 and 44 kDa RXRa are now under investigation in our laboratory. ACKNOWLEDGMENTS We are grateful to Dr. Greg Pratt and Professor Martin Rechsteiner, University of Utah, for kindly helping us use the PEST-FIND program on line.
REFERENCES 1. Levin, A. A., Sturzenbecker, L. J., Kazmer, S., Bosakowski, T., Huselton, C., Allenby, G., Speck, J., Kratzeisen, C., Rosenberger, M., Lovey, A., and Grippo, J. F. (1992) Nature 355, 359–361. 950
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2. Heyman, R. A., Mangelsdorf, D. J., Dyck, J. A., Stein, R. B., Eichele, G., Evans, R. M., and Thaller, C. (1992) Cell 68, 387–406. 3. Allenby, G., Bocquel, M.-T., Saunders, M., Kazmer, S., Speck, J., Rosenberger, M., Lovey, A., Kastner, P., Grippo, J. F., Chambon, P., and Levin, A. A. (1993) Proc. Natl. Acad. Sci. USA 90, 30–34. 4. Mangelsdorf, D. J., and Evans, R. M. (1995) Cell 83, 841–850. 5. Mangelsdorf, D. J., Umesono, K., and Evans, R. M. (1994) in The Retinoids, Biology, Chemistry, and Medicine, 2nd Ed. (Sporn, M. B., Roberts, A. B., and Goodman, D. S., Eds.), Raven Press, New York. 6. Mangelsdorf, D. J., Ong, E. S., Dyck, J. A., and Evans, R. M. (1990) Nature 345, 224–229. 7. Titcomb, M. W., Gottardis, M. M., Pike, J. W., and Allegretto, E. A. (1994) Mol. Endocrinol. 8, 870–877. 8. Yamada, Y., Shidoji, Y., Fukutomi, Y., Ishikawa, T., Kaneko, T., Nakagama, H., Imawari, M., Moriwaki, H., and Muto, Y. (1994) Mol. Carcinogenesis 10, 151–158. 9. Krust, A., Green, S., Argos, P., Kumar, V., Walter, P., Bornert, J. M., and Chambon, P. (1986) EMBO J. 5, 891– 897. 10. Bourguet, W., Ruff, M., Chambon, P., Gronemmeyer, H., and Moras, D. (1995) Nature 375, 377–382. 11. Nagpal, S., Saunders, M., Kastner, P., Durrand, B., Nakshatri, H., and Chambon, P. (1992) Cell 70, 1007–1019. 12. Nagpal, S., Friant, S., Nakshatri, H., and Chambon, P. (1993) EMBO J. 12, 2349–2360. 13. Matsushima-Nishwaki, R., Shidoji, Y., Nishiwaki, S., Yamada, T., Moriwaki, H., and Muto, Y. (1996) Mol. Cell. Endocrinol., in press. 14. Watt, F., and Molloy, P. L. (1993) Nucleic Acid Res. 21, 5092–5100. 15. Suzuki, K., and Ohno, S. (1990) Cell Structure and Function 15, 1–6. 16. Wang, K. K. W., Villalobo, A., and Roufogalis, B. D. (1989) Biochem. J. 262, 693–706. 17. Hirai, S., Kawasaki, H., Yaniv, M., and Suzuki, K. (1991) FEBS Lett. 287, 57–61. 18. Rochette-Egly, C., Lutz, Y., Saunders, M., Scheuer, I., Gaub, M.-P., and Chambon, P. (1991) J. Cell. Biol. 115, 535–545. 19. Tanaka, K., Sato, M., and Ichihara, A. (1978) J. Biochem. (Tokyo) 84, 937–946. 20. Bradford, M. M. (1970) Anal. Biochem. 72, 248–254. 21. Schollmeyer, J. E. (1988) Science 240, 911–913. 22. Lane, R. D., Allan, D. M., and Mellgren, R. L. (1992) Exp. Cell. Res. 203, 5–16. 23. Goll, D. E., Thompson, V. F., Taylor, R. G., and Zalewska, T. (1992) BioEssays 14, 549–556. 24. Mellgren, R. L., and Rozanov, C. B. (1990) Biochem. Biophys. Res. Commun. 168, 589–595. 25. Rogers, S., Well, R., and Rechsteiner, M. (1986) Science 234, 364–368. 26. Mader, S., Chen, J.-Y., Chen, J., White, J., Chambon, P., and Gronomeyer, H. (1993) EMBO J. 12, 5029–5041.
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Limited Degradation of Retinoid X Receptor by Calpain Rie Matsushima-Nishiwaki, Yoshihiro Shidoji, Shinji Nishiwaki, Hisataka Moriwaki, and Yasutoshi Muto1 First Department of Internal Medicine, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu city, Gifu 500, Japan Received July 15, 1996 Recently, we have found two major physiological forms of retinoid X receptor a (RXRa): the mature 54 kDa RXRa and the truncated 44 kDa RXRa lacking a portion of N-terminal A/B domain in human and rodent livers. In this communication, we show that m-calpain was active to digest 54 kDa RXRa in the human hepatoma-derived cell line, HuH7, nuclei to 44 kDa fragment through 47 kDa intermediate in vitro. Although both proteolytic fragments were revealed by anti-RXRa antibody against its E-domain, neither fragment reacted with anti-RXRa antibody specific for A/B domain. The profile of the calpaininduced proteolytic fragmentation of RXRa was almost identical to that of endogenous RXRa in nonmalignant human and normal mouse liver nuclei. This is the first demonstration that RXRa is a substrate for mcalpain, strongly suggesting that the enzyme might also be involved in post-translational modification of the receptor in hepatocytes. q 1996 Academic Press, Inc.
Retinoid X receptors (RXRs) belong to nuclear receptor superfamily, bind 9-cis retinoic acid as an endogenous ligand, and regulate several gene expressions (1-3). RXR forms homodimer and heterodimers with retinoic acid receptors, vitamin D receptor, thyroid hormone receptors, peroxisome proliferator-activated receptors or other orphan receptors (4) and is now recognized to play a central role in the biologic function of the nuclear receptor superfamily. These dimers bind their response elements and subsequently activate or inhibit the expression of their target genes (5). There are three RXR subtypes (a, b and g) and these subtypes show the different organ expression patterns (5). RXRa is the most abundant in the liver (6) and is also highly expressed in human hepatocellular carcinoma (HCC)-derived HuH7 cells (7, 8). RXRs are characterized by a modular domain structure and they contain the A through E regions (9). The non-conserved N-terminal A/B domain has an autonomous transcriptional activation function (AF-1), a fairly conserved C domain shows DNA binding activity, and beyond a hinge domain D, the variable C-terminal E domain gives ligand binding pocket (10) and dimerization activities with ligand dependent activation function (AF-2) (11, 12). Recently, we have found the two major physiological forms of the nuclear RXRa; the mature 54 kDa RXRa and the truncated 44 kDa RXRa lacking a portion of amino-terminal A/B domain in the human and mouse livers and, furthermore, we have shown that the intranuclear metabolism from 54 kDa to 44 kDa RXR was impaired in HCC compared with its surrounding non-malignant tissues (13). Even though we are still unaware of the functional aspect in the proteolytic fragmentation, it is intriguing for us to study the enzymatic regulation of molecular conversion from 54 kDa to 44 kDa RXRa in order to scrutinize irreversible posttranslational transformation of nuclear transcription factors in general. 1 To whom correspondence should be addressed. Fax: /81-58-262-8484. Abbreviations used: RXR, retinoid X receptor; HCC, human hepatocellular carcinoma; EDTA, ethylene diamine tetraacetic acid; SDS, sodium dodecyl sulfate; ECL, enhanced chemiluminescence; RXRE, retinoid X receptor response element.
946 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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In the literatures, studies on proteolytic cleavage of nuclear transcription factors are scanty, but, Watt and Molloy (14) have found that a number of sequence-specific transcription factors such as USF, AP-1, Pit-1, Oct-1, CP1a, c-Myc, CREB, AP-2 and AP-3, but not Sp-1 were limitedly cleaved by nonlysosomal calcium-dependent cysteine protease, m-calpain. The calpain is distributed in ubiquitous tissues and cells, but its substrates are relatively limited (15). Since these include nuclear hormone-receptors such as progesterone receptor or estrogen receptor (16) as well as other transcription factors (14, 17), we were very much interested to know whether m-calpain is involved in production of 44 kDa RXRa or not. In this communication, we show that the 54 kDa RXRa was in vitro digested at A/B domain into 44 kDa RXRa by m-calpain through 47 kDa intermediate. MATERIALS AND METHODS Preparation of nuclear extracts. Nuclear extracts were prepared from HuH7 cells with or without protease inhibitors (leupeptin, pepstatin A, chymostatin and a-1 antitrypsin) and also from mouse primary cultured hepatocytes, according to the method of Rochette-Egly et al (18) with minor modifications. Mouse hepatocytes were isolated from male CD1 mice livers digested with collagenase (19) and cultured on a plastic flask. Determination of protein concentrations was conducted according to the method of Bradford (20). Digestion of the nuclear extracts with m-calpain. m-Calpain was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Digestion was conducted according to the method of Hirai et al (17). Briefly, the nuclear extracts of HuH7 cells were mixed on ice with a calpain solution (5 ml), and the reaction was started by adding 5 ml of the reaction mixture (0.4 M Tris-HCl, pH 7.8, 12 mM CaCl2 , and 2 mM dithiothreithol) at 307C. In some experiments, CaCl2 in reaction mixture was replaced with 12 mM ethylene diamine tetraacetic acid (EDTA). After incubation for 10 min., 4% sodium dodecyl sulfate (SDS) sample buffer (20 ml) was added and boiled for 5 min to stop the reaction. Western blot analysis. The boiled samples were electrophoresed on a 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane. The immunodetection was performed with polyclonal anti-RXRa(E) or D-20. Polyclonal anti-RXRa(E) was raised against E domain of the recombinant human RXRa as described elsewhere (13), and D-20, the polyclonal antibody for A/B domain of human RXRa was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). And the membrane was submitted to the enhanced chemiluminescence (ECL) detection, according to the protocol provided by Amersham, Buckinghamshire, UK. After exposure of the blotting membrane to an X-ray film, the film was scanned by JX-330M scanner (Sharp, Osaka, Japan) and densitometric analyses were conducted by using the NIH Image version 1.59 software. PEST sequence analysis. PEST sequence analysis was performed using PEST sequence searching program provided through Internet (http://www.biu.icnet.uk/projects/pest/) by Dr. Rechsteiner, University of Utah.
RESULTS AND DISCUSSION
As shown in Figure 1A, the major form of RXRa in human HCC-derived HuH7 cells was 54 kDa, predicted size from cDNA sequence of RXRa and minor bands of 47 kDa and 44 kDa were also detected. In a sharp contrast, the major form of RXRa in normal mouse primary cultured hepatocytes was found as 44 kDa with a faint band of 54 kDa RXRa, even-though a cocktail of protease inhibitors were included during the preparation of the nuclear extracts. The 44 kDa RXRa of mouse hepatocytes as well as 47 kDa and 44 kDa RXRa of HuH7 cells did not react with D-20, anti-A/B domain of RXRa, whereas the antibody detected 54 kDa species on the same filter (Figure 1B). This would suggest that the both 47 kDa and 44 kDa RXRa lack a portion of A/B domain and are the intranuclear proteolytic fragments of the mature 54 kDa RXRa. The previous reports on proteases responsible for proteolytic cleavage of nuclear receptors were limited, but progesterone receptor and estrogen receptor are substrates for m-calpain which associates subcellular organelles including the nuclear fraction (16, 21, 22). Although, it is an isozyme that requires a calcium concentration of ú0.2 mM for full activity in vitro (23), it was found to reach half maximal activity as 3 mM Ca2/ in isolated rat liver nuclei (24). Therefore, we prepared the nuclear extracts of HuH7 cells in the absence of protease inhibitors and then added exogenous m-calpain to the extracts. Compared with lane 1 in Figure 1A, a slight increase in 47 kDa and 44 kDa RXRa was already found in the nuclear extracts 947
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FIG. 1. Western blotting analyses of the intranuclear molecular species of RXRa. The nuclear extracts prepared with protease inhibitors from human hepatocellular carcinoma derived HuH7 cells (lanes 1) and from mouse primary cultured hepatocytes (lanes 2) were submitted to immunoblotting using anti-hRXRa(E) (panel A) and D-20, antiA/B domain of hRXRa (panel B). Arrow indicates the mobility of 54 kDa, arrow with dotted line indicates that of 47 kDa, and arrowhead indicates that of 44 kDa.
incubated with 3 mM Ca2/ and without protease inhibitors (Figure 2A, lane 1), suggesting that the nuclear extracts may contain endogenous Ca2/-activated protease activity. Exogenously added m-calpain attacked 54 kDa RXRa as well as 47 kDa RXRa, but 44 kDa RXRa was quite stable even in the presence of 100 mg/ml calpain (Figure 2A, lane 5). The amount of 54 kDa RXRa decreased in accordance with the increase of 47 kDa RXRa in the concentration dependent manner of m-calpain added. m-Calpain cleaved almost all of the 54 and 47 kDa RXRa into 44 kDa RXRa at the concentration of 100 mg/ml. In the absence of the Ca2/, the proportion of the 54, 47 and 44 kDa RXRa in the nuclear extract of HuH7 cells was not
FIG. 2. Concentration dependence of m-calpain-induced proteolytic profiles of RXRa in the nuclear extracts. (A) The nuclear extracts from HuH7 cells, prepared without protease inhibitors, were incubated with the indicated concentrations of m-calpain in the presence or absence of Ca2/. After 10 min incubation at 307C, the digests were separated in a 10% SDS-polyacrylamide gel and the RXRas were detected by immunoblotting with anti-hRXRa(E). (B) Percent distribution of each RXRa species to total RXRa in each lane of (A) was calculated after densitmetric analysis. 54 kDa (l), 47 kDa (n), 44 kDa (s). 948
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FIG. 3. Lack of reactivity with anti-A/B domain antibody in the calpain-induced fragments of RXRa. RXRa was treated with m-calpain in the presence (lanes 3) or absence (lanes 2) of Ca2/ and submitted to immunoblotting using anti-hRXRa(E) (panel A) or D-20, anti-A/B domain of RXRa (panel B).
changed even at 100 mg/ml m-calpain (Figure 2A). Thus, the digestion from 54 kDa RXRa to 47 kDa and 44 kDa fragment was absolutely Ca2/-dependent, excluding the possibility that other protease activity contaminated in m-calpain preparation may digest 54 kDa RXRa. The densitometric analysis (Figure 2B) clearly shows that 47 kDa RXRa was an intermediate form produced on the way of the digestion from 54 kDa RXRa to 44 kDa. As mentioned above, the physiologically truncated forms of 47 and 44 kDa RXRa lacked a portion of A/B domain. The molecular sizes of m-calpain-induced products were almost identical to those of the physiological fragments of RXRa, however, we had to determine whether the calpain-products were lacking in A/B domain from the 54 kDa mature RXRa of the nuclear extract of HuH7 cells. As shown in Figure 3, the 44 kDa RXRa which reacted with the anti-RXRa(E) antibody failed to react with D-20 antibody, so did the 47 kDa RXRa under the condition where the antibody detected 54 kDa RXRa. Therefore, the 44 and 47 kDa RXRa are supposed to be digested from 54 kDa RXRa by m-calpain at A/B domain, and are relevant to the physiologically truncated forms of RXRa. It has been proposed that the amino acid sequences of most short-lived proteins include one or more regions rich in proline (P), glutamic acid (E), aspartic acid, serine (S) and threonine (T). These regions, known as ‘PEST’ regions, may be recognized directly or indirectly by calpain, which hydrolyzes a peptide bond at the vicinity of PEST region (25). The sequence of the PEST region of RXRs were searched by using the PEST-FIND program provided through Internet by Dr. Rechsteiner, University of Utah, UT, USA (25) (Figure 4). Two potential PEST sequences were found at amino acids 75-108 mapping at carboxy terminus half of A/B domain and 215-234 mapping at carboxy terminus half of hinge D domain of human, mouse and rat RXRa. One can reasonably speculate that m-calpain may digest at the vicinity of the first potential PEST region resided on A/B domain of 54 kDa RXRa to produce 47 kDa RXRa, leaving DNA-binding C domain intact. The sequence of amino acids 75-108 was highly conserved between RXR subtypes. The same region in A/B domain in RXRb was also the potential PEST sequence, and in RXRg, it was poor but candidate PEST sequence. 949
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FIG. 4. Diagrammatic representation of PEST regions in the amino acid sequences of RXRas, RXRbs, and RXRg. The possible PEST regions (0 õ PEST-FIND score) are indicated by closed boxes, and the poor candidate PEST regions (0 § PEST-FIND score) are indicated by open boxes. PEST regions were searched by PEST-FIND program as described in Materials and Methods.
To produce 44 kDa RXRa from 47 kDa RXRa, a candidate PEST sequence of amino acids 440-459 mapping at amino terminus of human RXRa is the most probable site for digestion. Even though the region is poor candidate PEST sequence, the most carboxy terminal region called tc is three dimensionally extended to outside from the core structure of the RXRa, thus it must be easily accessible to the protease. The most carboxy terminal region of the the RXRs is also highly conserved, and the regions in RXRb and g were also poor but candidate PEST sequence. This carboxy terminus region is very important position for the ligand dependent transcription, named as AF-2 (4). Another ligand independent transcription mediated region, AF-1 is located at A/B domain of RXRs (4). Taking account of these functional modular domains of RXR, the present results suggest that RXRa may lose AF-1 at the initial step of the proteolysis and thereafter lose AF-2 at the second step from 47 kDa to 44 kDa digestion by m-calpain, and these proteolyses may be happened to not only RXRa but also to b and g. According to the report that the recombinant RXRDAB, lacking in A/B domain, hetero- or homo-dimerized and binded to synthetic direct repeats (26), 47 kDa and 44 kDa RXR found in calpain-digests may well bind to natural response elements. In conclusion, m-calpain cleaved out an N-terminal portion of A/B domain from the mature RXRa to produce 47 kDa and 44 kDa fragments. This unique irreversible two-step transformation of RXRa might be important for the signal transduction of retinoids. The exact digestion sites and the role of the 47 and 44 kDa RXRa are now under investigation in our laboratory. ACKNOWLEDGMENTS We are grateful to Dr. Greg Pratt and Professor Martin Rechsteiner, University of Utah, for kindly helping us use the PEST-FIND program on line.
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