The Linkage of Kennedy's Neuron Disease to ARA24, the First Identified Androgen Receptor Polyglutamine Region-associated Coactivator

Abstract

Although the linkage of polyglutamine (poly-Q) repeat expansion in the androgen receptor (AR) to Kennedy's disease (X-linked spinal and bulbar muscular atrophy) was a major step forward, the detailed molecular mechanism of how the change in poly-Q length contributes to the disease remains unclear. Here we report the identification of a nuclear G-protein, Ras-related nuclear protein/ARA24, as the first AR coactivator that can bind differentially with different lengths of poly-Q within AR. In the yeast and mammalian reciprocal interacting assays, our data suggested the interaction of AR N-terminal domain with ARA24 diminishes as the poly-Q length increases. The coactivation of ARA24 also diminishes with the poly-Q expansion within AR. Deletion of the acidic hexapeptide (DEDDDL) at the C terminus of ARA24 further enhances its AR coactivation. Together, our data suggest that poor interaction and weaker coactivation of ARA24 to the longer poly-Q AR in the X-linked spinal and bulbar muscular atrophied AR could contribute to the weaker transactivation of AR. The consequence of poor interaction and weak coactivation may eventually lead to the partial androgen insensitivity during the development of Kennedy's disease. Although the linkage of polyglutamine (poly-Q) repeat expansion in the androgen receptor (AR) to Kennedy's disease (X-linked spinal and bulbar muscular atrophy) was a major step forward, the detailed molecular mechanism of how the change in poly-Q length contributes to the disease remains unclear. Here we report the identification of a nuclear G-protein, Ras-related nuclear protein/ARA24, as the first AR coactivator that can bind differentially with different lengths of poly-Q within AR. In the yeast and mammalian reciprocal interacting assays, our data suggested the interaction of AR N-terminal domain with ARA24 diminishes as the poly-Q length increases. The coactivation of ARA24 also diminishes with the poly-Q expansion within AR. Deletion of the acidic hexapeptide (DEDDDL) at the C terminus of ARA24 further enhances its AR coactivation. Together, our data suggest that poor interaction and weaker coactivation of ARA24 to the longer poly-Q AR in the X-linked spinal and bulbar muscular atrophied AR could contribute to the weaker transactivation of AR. The consequence of poor interaction and weak coactivation may eventually lead to the partial androgen insensitivity during the development of Kennedy's disease. Kennedy's disease (SBMA) 1The abbreviations SBMAX-linked spinal and bulbar muscular atrophyARandrogen receptorpoly-QpolyglutamineARA24AR-associated protein 24RanRas-related nuclear proteinRCC1regulator of chromosome condensation 1ARA24asantisense ARA24ARA160AR-associated protein 160LucluciferaseDHT5α-dihydrotestosteroneCATchloramphenicol acetyltransferaseGBDGAL4 DNA binding domainWTwild type 1The abbreviations SBMAX-linked spinal and bulbar muscular atrophyARandrogen receptorpoly-QpolyglutamineARA24AR-associated protein 24RanRas-related nuclear proteinRCC1regulator of chromosome condensation 1ARA24asantisense ARA24ARA160AR-associated protein 160LucluciferaseDHT5α-dihydrotestosteroneCATchloramphenicol acetyltransferaseGBDGAL4 DNA binding domainWTwild typeis a motor neuron disease characterized by progressive muscle weakness and atrophy (1Kennedy W.R. Alter M. Sung J.H. Neurology. 1968; 18: 671-680Crossref PubMed Google Scholar). Over 50% of the affected males may also have gynecomastia and reduced fertility, suggesting a defect in AR function (2Nagashima T. Seko K. Hirose K. Mannen T. Yoshimura R. Arima R. Nagashima K. Morimatsu Y. J. Neurol. Sci. 1988; 87: 141-152Abstract Full Text PDF PubMed Scopus (80) Google Scholar). This hypothesis was confirmed by finding an expansion of poly-Q in SBMA AR (3La Spada A.R. Wilson E.M. Lubahn D.B. Harding A.E. Fischbeck K.H. Nature. 1991; 352: 77-79Crossref PubMed Scopus (2380) Google Scholar). Several hypotheses have been proposed to explain how changes in poly-Q length within AR contribute to the disease. 1) SBMA AR may reduce binding affinity to androgen (4MacLean H.E. Choi W.T. Rekaris G. Warne G.L. Zajac J.D. J. Clin. Endocrinol. Metab. 1995; 80: 508-516Crossref PubMed Google Scholar); 2) SBMA AR may become more unstable and can degrade to small toxic AR (5Choong C.S. Kemppainen J.A. Zhou Z.X. Wilson E.M. Mol. Endocrinol. 1996; 10: 1527-1535Crossref PubMed Scopus (265) Google Scholar); and 3) SBMA AR may aggregate in cytoplasm by cross-linking with itself or other proteins (6Merry D.E. Kobayashi Y. Bailey C.K. Taye A.A. Fischbeck K.H. Hum. Mol. Genet. 1998; 7: 693-701Crossref PubMed Scopus (165) Google Scholar). The last hypothesis inspired us to find the potential AR-associated proteins that may contribute to this disease. Using the yeast two-hybrid system we were able to find an AR coactivator, ARA24, that can differentially activate the AR with different poly-Q lengths within AR. Nucleotide sequencing revealed that this AR coactivator has an identical sequence with Ran (7Bischoff F.R. Ponstingl H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10830-10834Crossref PubMed Scopus (220) Google Scholar).Genetic results have identified that Ran/ARA24 is involved in nuclear transport of proteins and RNA, cell cycle progression, and nuclear structure in mitotic regulation, as well as RNA and DNA synthesis (8Rush M.G. Drivas G. D'Eustachio P. Bioessays. 1996; 18: 103-112Crossref PubMed Scopus (91) Google Scholar). Ran/ARA24 was purified as a complex with a chromatin-associated DNA-binding protein, RCC1 (regulator of chromosome condensation 1). RCC1 was first identified as the product of a gene mutated in the tsBN2 (temperature-sensitive baby hamster kidney) cell line (9Nishitani H. Ohtsubo M. Yamashita K. Iida H. Pines J. Yasudo H. Shibata Y. Hunter T. Nishimoto T. EMBO J. 1991; 10: 1555-1564Crossref PubMed Scopus (151) Google Scholar). RCC1 has been shown to function as a guanine nucleotide exchange factor on Ran/ARA24 by increasing its rate ∼5 × 105 times. On the other hand, Ran GTPase-activating protein can also increase the GTPase activity ∼1 × 105 times (10Klebe C. Bischoff F.R. Ponstingl H. Wittinghofer A. Biochemistry. 1995; 34: 639-647Crossref PubMed Scopus (267) Google Scholar). During biochemical identification of Ran-associated protein, previous reports mainly revealed its functions in nuclear transport (8Rush M.G. Drivas G. D'Eustachio P. Bioessays. 1996; 18: 103-112Crossref PubMed Scopus (91) Google Scholar). Here we demonstrate that Ran/ARA24 can also interact with the AR N-terminal poly-Q region and enhance the AR transactivation.DISCUSSIONPrevious studies concerning the transactivation activity of AR with different poly-Q lengths have consistently shown their inverse relationship (21Yeh S. Lin H. Kang H.-Y. Lin M. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1998; 96: 5458-5463Crossref Scopus (498) Google Scholar, 25Kazemi-Esfarjani P. Trifiro M.A. Pinsky L. Hum. Mol. Genet. 1995; 4: 523-527Crossref PubMed Scopus (381) Google Scholar). The present study demonstrates that ARA24 interacts with the AR poly-Q region and functions as an AR coactivator. The interaction between AR and ARA24 decreases with the expansion of AR poly-Q, as well as the coactivation of cotransfected ARA24 to the SBMA AR. These results provide a potential mechanism for the partial androgen-insensitive syndrome in Kennedy's disease.It is well documented that a continual cycling of binding and hydrolyzing GTP from ARA24 is required in multiple cellular processes (reviewed in Ref. 8Rush M.G. Drivas G. D'Eustachio P. Bioessays. 1996; 18: 103-112Crossref PubMed Scopus (91) Google Scholar). Yeast mutations prp20 andrna1, which are homologues of mammalian Ran-guanine nucleotide exchange factor and Ran-GTPase-activating protein, respectively, have been shown to exhibit the phenotype of reduced accuracy in positioning the correct transcription initiation site (26Forrester W. Stutz F. Rosbash M. Wickens M. Genes Dev. 1992; 6: 1914-1926Crossref PubMed Scopus (112) Google Scholar). These phenotypes are readily explained by either a change in chromatin structure or by certain defects in the transcription regulation. The ability to enhance AR transactivation by ARA24 may therefore provide a bridge for ARA24 to link to the transcriptional regulation.As shown in Fig. 1 B, AR forms complexes with ARA24 regardless of the bound guanine nucleotide, which seems different from what has been observed for a GTPase effector protein that exhibits selective binding to either a GTPase-GTP or a GTPase-GDP. Upon inactivation of RCC1, ARA24 was shown dispersed in the nucleus and cytosol, suggesting that GTP-bound ARA24 is the major form in the nucleus (27Ren M.G. Drivas P. D'Eustachio P. Rush M.G. J. Cell Biol. 1993; 120: 313-323Crossref PubMed Scopus (153) Google Scholar). It seems that the AR-ARA24 interaction is not regulated by the guanine binding status of ARA24 in vitro. It is not clear whether the guanine binding status of ARA24 plays any role when mediating AR transactivation in cells.ARA24 is a very abundant cellular protein. It is estimated that HeLa cells contain about 107 ARA24 molecules per cell (7Bischoff F.R. Ponstingl H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10830-10834Crossref PubMed Scopus (220) Google Scholar). This high abundance does cause great difficulty when attempting to show its biochemical function by the addition of exogenous ARA24. This is probably the reason we need to use a relatively high dose of exogenous ARA24 to show its coactivator activity (Fig. 2 A). Nevertheless, the ratios of receptor to coactivator we used here (1:10 or 1:30) are close to the ratios of other steroid receptors to their coactivators. For example, full-length SRC-1 requires the addition ratio of 1:20 or 1:40 to see the coactivator effects (28Takeshita A. Cardona G.R. Koibuchi N. Suen C.-S. Chin W.W. J. Biol. Chem. 1997; 272: 27629-27634Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). On the other hand, cotransfection of ARA24as to decrease the cellular ARA24 protein level can also repress the AR transactivation. As shown in Fig.2 B, deleting the six amino acid residues of the acidic C terminus in ARA24 results in a dominant positive AR coactivator. A lower (500 ng) level of ARA24 is to a coactivation to AR with of ARA24. increasing ARA24 to a dose ng) further enhances AR transactivation from to As the hexapeptide sequence of ARA24 is is that a factor may bind to the hexapeptide and of this hexapeptide may therefore ARA24 to function more as a coactivator to enhance AR AR coactivator effects of ARA24 and are not of AR transactivation at the of also at the level that is in androgen The increase of androgen by the could of the the level increase the is that either ARA24 or can increase androgen that can androgen may provide molecular mechanism to explain the of androgen poly-Q of AR has been for with Kennedy's disease. studies also show that the longer poly-Q may in weaker AR transactivation (21Yeh S. Lin H. Kang H.-Y. Lin M. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1998; 96: 5458-5463Crossref Scopus (498) Google Scholar, 25Kazemi-Esfarjani P. Trifiro M.A. Pinsky L. Hum. Mol. Genet. 1995; 4: 523-527Crossref PubMed Scopus (381) Google Scholar). have further the poly-Q AR with and a of M. 1997; Google Scholar, K. M. A. J. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). results that ARA24 can interact differentially with different lengths of poly-Q within and its ability to enhance AR transactivation also decreases with expansion of poly-Q length in AR. A very to in the whether of ARA24 or its ability to interact with the AR poly-Q region could become an to ARA24 is also involved in nuclear that can the nuclear of is that the weaker interaction of ARA24 to longer poly-Q AR may in of SBMA AR in of the nucleus to function as a transcriptional A detailed of of ARA24 and SBMA AR in Kennedy's disease cells able to an our results suggested that ARA24 is the first identified AR N-terminal coactivator that can interact differentially with AR different poly-Q This finding may us to further the potential of AR function in Kennedy's disease and of Kennedy's disease (SBMA) 1The abbreviations SBMAX-linked spinal and bulbar muscular atrophyARandrogen receptorpoly-QpolyglutamineARA24AR-associated protein 24RanRas-related nuclear proteinRCC1regulator of chromosome condensation 1ARA24asantisense ARA24ARA160AR-associated protein 160LucluciferaseDHT5α-dihydrotestosteroneCATchloramphenicol acetyltransferaseGBDGAL4 DNA binding domainWTwild type 1The abbreviations SBMAX-linked spinal and bulbar muscular atrophyARandrogen receptorpoly-QpolyglutamineARA24AR-associated protein 24RanRas-related nuclear proteinRCC1regulator of chromosome condensation 1ARA24asantisense ARA24ARA160AR-associated protein 160LucluciferaseDHT5α-dihydrotestosteroneCATchloramphenicol acetyltransferaseGBDGAL4 DNA binding domainWTwild typeis a motor neuron disease characterized by progressive muscle weakness and atrophy (1Kennedy W.R. Alter M. Sung J.H. Neurology. 1968; 18: 671-680Crossref PubMed Google Scholar). Over 50% of the affected males may also have gynecomastia and reduced fertility, suggesting a defect in AR function (2Nagashima T. Seko K. Hirose K. Mannen T. Yoshimura R. Arima R. Nagashima K. Morimatsu Y. J. Neurol. Sci. 1988; 87: 141-152Abstract Full Text PDF PubMed Scopus (80) Google Scholar). This hypothesis was confirmed by finding an expansion of poly-Q in SBMA AR (3La Spada A.R. Wilson E.M. Lubahn D.B. Harding A.E. Fischbeck K.H. Nature. 1991; 352: 77-79Crossref PubMed Scopus (2380) Google Scholar). Several hypotheses have been proposed to explain how changes in poly-Q length within AR contribute to the disease. 1) SBMA AR may reduce binding affinity to androgen (4MacLean H.E. Choi W.T. Rekaris G. Warne G.L. Zajac J.D. J. Clin. Endocrinol. Metab. 1995; 80: 508-516Crossref PubMed Google Scholar); 2) SBMA AR may become more unstable and can degrade to small toxic AR (5Choong C.S. Kemppainen J.A. Zhou Z.X. Wilson E.M. Mol. Endocrinol. 1996; 10: 1527-1535Crossref PubMed Scopus (265) Google Scholar); and 3) SBMA AR may aggregate in cytoplasm by cross-linking with itself or other proteins (6Merry D.E. Kobayashi Y. Bailey C.K. Taye A.A. Fischbeck K.H. Hum. Mol. Genet. 1998; 7: 693-701Crossref PubMed Scopus (165) Google Scholar). The last hypothesis inspired us to find the potential AR-associated proteins that may contribute to this disease. Using the yeast two-hybrid system we were able to find an AR coactivator, ARA24, that can differentially activate the AR with different poly-Q lengths within AR. Nucleotide sequencing revealed that this AR coactivator has an identical sequence with Ran (7Bischoff F.R. Ponstingl H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10830-10834Crossref PubMed Scopus (220) Google Scholar). X-linked spinal and bulbar muscular atrophy androgen receptor polyglutamine AR-associated protein Ras-related nuclear protein of chromosome condensation 1 ARA24 AR-associated protein DNA binding domain type X-linked spinal and bulbar muscular atrophy androgen receptor polyglutamine AR-associated protein Ras-related nuclear protein of chromosome condensation 1 ARA24 AR-associated protein DNA binding domain type results have identified that Ran/ARA24 is involved in nuclear transport of proteins and RNA, cell cycle progression, and nuclear structure in mitotic regulation, as well as RNA and DNA synthesis (8Rush M.G. Drivas G. D'Eustachio P. Bioessays. 1996; 18: 103-112Crossref PubMed Scopus (91) Google Scholar). Ran/ARA24 was purified as a complex with a chromatin-associated DNA-binding protein, RCC1 (regulator of chromosome condensation 1). RCC1 was first identified as the product of a gene mutated in the tsBN2 (temperature-sensitive baby hamster kidney) cell line (9Nishitani H. Ohtsubo M. Yamashita K. Iida H. Pines J. Yasudo H. Shibata Y. Hunter T. Nishimoto T. EMBO J. 1991; 10: 1555-1564Crossref PubMed Scopus (151) Google Scholar). RCC1 has been shown to function as a guanine nucleotide exchange factor on Ran/ARA24 by increasing its rate ∼5 × 105 times. On the other hand, Ran GTPase-activating protein can also increase the GTPase activity ∼1 × 105 times (10Klebe C. Bischoff F.R. Ponstingl H. Wittinghofer A. Biochemistry. 1995; 34: 639-647Crossref PubMed Scopus (267) Google Scholar). During biochemical identification of Ran-associated protein, previous reports mainly revealed its functions in nuclear transport (8Rush M.G. Drivas G. D'Eustachio P. Bioessays. 1996; 18: 103-112Crossref PubMed Scopus (91) Google Scholar). Here we demonstrate that Ran/ARA24 can also interact with the AR N-terminal poly-Q region and enhance the AR transactivation. studies concerning the transactivation activity of AR with different poly-Q lengths have consistently shown their inverse relationship (21Yeh S. Lin H. Kang H.-Y. Lin M. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1998; 96: 5458-5463Crossref Scopus (498) Google Scholar, 25Kazemi-Esfarjani P. Trifiro M.A. Pinsky L. Hum. Mol. Genet. 1995; 4: 523-527Crossref PubMed Scopus (381) Google Scholar). The present study demonstrates that ARA24 interacts with the AR poly-Q region and functions as an AR coactivator. The interaction between AR and ARA24 decreases with the expansion of AR poly-Q, as well as the coactivation of cotransfected ARA24 to the SBMA AR. These results provide a potential mechanism for the partial androgen-insensitive syndrome in Kennedy's disease.It is well documented that a continual cycling of binding and hydrolyzing GTP from ARA24 is required in multiple cellular processes (reviewed in Ref. 8Rush M.G. Drivas G. D'Eustachio P. Bioessays. 1996; 18: 103-112Crossref PubMed Scopus (91) Google Scholar). Yeast mutations prp20 andrna1, which are homologues of mammalian Ran-guanine nucleotide exchange factor and Ran-GTPase-activating protein, respectively, have been shown to exhibit the phenotype of reduced accuracy in positioning the correct transcription initiation site (26Forrester W. Stutz F. Rosbash M. Wickens M. Genes Dev. 1992; 6: 1914-1926Crossref PubMed Scopus (112) Google Scholar). These phenotypes are readily explained by either a change in chromatin structure or by certain defects in the transcription regulation. The ability to enhance AR transactivation by ARA24 may therefore provide a bridge for ARA24 to link to the transcriptional regulation.As shown in Fig. 1 B, AR forms complexes with ARA24 regardless of the bound guanine nucleotide, which seems different from what has been observed for a GTPase effector protein that exhibits selective binding to either a GTPase-GTP or a GTPase-GDP. Upon inactivation of RCC1, ARA24 was shown dispersed in the nucleus and cytosol, suggesting that GTP-bound ARA24 is the major form in the nucleus (27Ren M.G. Drivas P. D'Eustachio P. Rush M.G. J. Cell Biol. 1993; 120: 313-323Crossref PubMed Scopus (153) Google Scholar). It seems that the AR-ARA24 interaction is not regulated by the guanine binding status of ARA24 in vitro. It is not clear whether the guanine binding status of ARA24 plays any role when mediating AR transactivation in cells.ARA24 is a very abundant cellular protein. It is estimated that HeLa cells contain about 107 ARA24 molecules per cell (7Bischoff F.R. Ponstingl H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10830-10834Crossref PubMed Scopus (220) Google Scholar). This high abundance does cause great difficulty when attempting to show its biochemical function by the addition of exogenous ARA24. This is probably the reason we need to use a relatively high dose of exogenous ARA24 to show its coactivator activity (Fig. 2 A). Nevertheless, the ratios of receptor to coactivator we used here (1:10 or 1:30) are close to the ratios of other steroid receptors to their coactivators. For example, full-length SRC-1 requires the addition ratio of 1:20 or 1:40 to see the coactivator effects (28Takeshita A. Cardona G.R. Koibuchi N. Suen C.-S. Chin W.W. J. Biol. Chem. 1997; 272: 27629-27634Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). On the other hand, cotransfection of ARA24as to decrease the cellular ARA24 protein level can also repress the AR transactivation. As shown in Fig.2 B, deleting the six amino acid residues of the acidic C terminus in ARA24 results in a dominant positive AR coactivator. A lower (500 ng) level of ARA24 is to a coactivation to AR with of ARA24. increasing ARA24 to a dose ng) further enhances AR transactivation from to As the hexapeptide sequence of ARA24 is is that a factor may bind to the hexapeptide and of this hexapeptide may therefore ARA24 to function more as a coactivator to enhance AR AR coactivator effects of ARA24 and are not of AR transactivation at the of also at the level that is in androgen The increase of androgen by the could of the the level increase the is that either ARA24 or can increase androgen that can androgen may provide molecular mechanism to explain the of androgen poly-Q of AR has been for with Kennedy's disease. studies also show that the longer poly-Q may in weaker AR transactivation (21Yeh S. Lin H. Kang H.-Y. Lin M. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1998; 96: 5458-5463Crossref Scopus (498) Google Scholar, 25Kazemi-Esfarjani P. Trifiro M.A. Pinsky L. Hum. Mol. Genet. 1995; 4: 523-527Crossref PubMed Scopus (381) Google Scholar). have further the poly-Q AR with and a of M. 1997; Google Scholar, K. M. A. J. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). results that ARA24 can interact differentially with different lengths of poly-Q within and its ability to enhance AR transactivation also decreases with expansion of poly-Q length in AR. A very to in the whether of ARA24 or its ability to interact with the AR poly-Q region could become an to ARA24 is also involved in nuclear that can the nuclear of is that the weaker interaction of ARA24 to longer poly-Q AR may in of SBMA AR in of the nucleus to function as a transcriptional A detailed of of ARA24 and SBMA AR in Kennedy's disease cells able to an our results suggested that ARA24 is the first identified AR N-terminal coactivator that can interact differentially with AR different poly-Q This finding may us to further the potential of AR function in Kennedy's disease and of studies concerning the transactivation activity of AR with different poly-Q lengths have consistently shown their inverse relationship (21Yeh S. Lin H. Kang H.-Y. Lin M. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1998; 96: 5458-5463Crossref Scopus (498) Google Scholar, 25Kazemi-Esfarjani P. Trifiro M.A. Pinsky L. Hum. Mol. Genet. 1995; 4: 523-527Crossref PubMed Scopus (381) Google Scholar). The present study demonstrates that ARA24 interacts with the AR poly-Q region and functions as an AR coactivator. The interaction between AR and ARA24 decreases with the expansion of AR poly-Q, as well as the coactivation of cotransfected ARA24 to the SBMA AR. These results provide a potential mechanism for the partial androgen-insensitive syndrome in Kennedy's disease. It is well documented that a continual cycling of binding and hydrolyzing GTP from ARA24 is required in multiple cellular processes (reviewed in Ref. 8Rush M.G. Drivas G. D'Eustachio P. Bioessays. 1996; 18: 103-112Crossref PubMed Scopus (91) Google Scholar). Yeast mutations prp20 andrna1, which are homologues of mammalian Ran-guanine nucleotide exchange factor and Ran-GTPase-activating protein, respectively, have been shown to exhibit the phenotype of reduced accuracy in positioning the correct transcription initiation site (26Forrester W. Stutz F. Rosbash M. Wickens M. Genes Dev. 1992; 6: 1914-1926Crossref PubMed Scopus (112) Google Scholar). These phenotypes are readily explained by either a change in chromatin structure or by certain defects in the transcription regulation. The ability to enhance AR transactivation by ARA24 may therefore provide a bridge for ARA24 to link to the transcriptional regulation. As shown in Fig. 1 B, AR forms complexes with ARA24 regardless of the bound guanine nucleotide, which seems different from what has been observed for a GTPase effector protein that exhibits selective binding to either a GTPase-GTP or a GTPase-GDP. Upon inactivation of RCC1, ARA24 was shown dispersed in the nucleus and cytosol, suggesting that GTP-bound ARA24 is the major form in the nucleus (27Ren M.G. Drivas P. D'Eustachio P. Rush M.G. J. Cell Biol. 1993; 120: 313-323Crossref PubMed Scopus (153) Google Scholar). It seems that the AR-ARA24 interaction is not regulated by the guanine binding status of ARA24 in vitro. It is not clear whether the guanine binding status of ARA24 plays any role when mediating AR transactivation in ARA24 is a very abundant cellular protein. It is estimated that HeLa cells contain about 107 ARA24 molecules per cell (7Bischoff F.R. Ponstingl H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10830-10834Crossref PubMed Scopus (220) Google Scholar). This high abundance does cause great difficulty when attempting to show its biochemical function by the addition of exogenous ARA24. This is probably the reason we need to use a relatively high dose of exogenous ARA24 to show its coactivator activity (Fig. 2 A). Nevertheless, the ratios of receptor to coactivator we used here (1:10 or 1:30) are close to the ratios of other steroid receptors to their coactivators. For example, full-length SRC-1 requires the addition ratio of 1:20 or 1:40 to see the coactivator effects (28Takeshita A. Cardona G.R. Koibuchi N. Suen C.-S. Chin W.W. J. Biol. Chem. 1997; 272: 27629-27634Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). On the other hand, cotransfection of ARA24as to decrease the cellular ARA24 protein level can also repress the AR transactivation. As shown in Fig.2 B, deleting the six amino acid residues of the acidic C terminus in ARA24 results in a dominant positive AR coactivator. A lower (500 ng) level of ARA24 is to a coactivation to AR with of ARA24. increasing ARA24 to a dose ng) further enhances AR transactivation from to As the hexapeptide sequence of ARA24 is is that a factor may bind to the hexapeptide and of this hexapeptide may therefore ARA24 to function more as a coactivator to enhance AR transactivation. The AR coactivator effects of ARA24 and are not of AR transactivation at the of also at the level that is in androgen The increase of androgen by the could of the the level increase the is that either ARA24 or can increase androgen that can androgen may provide molecular mechanism to explain the of androgen The poly-Q of AR has been for with Kennedy's disease. studies also show that the longer poly-Q may in weaker AR transactivation (21Yeh S. Lin H. Kang H.-Y. Lin M. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1998; 96: 5458-5463Crossref Scopus (498) Google Scholar, 25Kazemi-Esfarjani P. Trifiro M.A. Pinsky L. Hum. Mol. Genet. 1995; 4: 523-527Crossref PubMed Scopus (381) Google Scholar). have further the poly-Q AR with and a of M. 1997; Google Scholar, K. M. A. J. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). results that ARA24 can interact differentially with different lengths of poly-Q within and its ability to enhance AR transactivation also decreases with expansion of poly-Q length in AR. A very to in the whether of ARA24 or its ability to interact with the AR poly-Q region could become an to As ARA24 is also involved in nuclear that can the nuclear of is that the weaker interaction of ARA24 to longer poly-Q AR may in of SBMA AR in of the nucleus to function as a transcriptional A detailed of of ARA24 and SBMA AR in Kennedy's disease cells able to an In our results suggested that ARA24 is the first identified AR N-terminal coactivator that can interact differentially with AR different poly-Q This finding may us to further the potential of AR function in Kennedy's disease and of