FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2106571316> ?p ?o ?g. }

• W2106571316 endingPage "7346" @default.
• W2106571316 startingPage "7341" @default.
• W2106571316 abstract "To locate in detail the regions in the human androgen receptor (AR) involved in transcription activation, a series of N-terminal deletions was introduced in the wild type AR and in a constitutively active AR. The different constructs were tested for their capacity to activate transcription. Almost the entire N-terminal domain (residues 1-485) was necessary for full wild type AR activity when cotransfected with the (GRE)2tkCAT reporter in HeLa cells. In contrast, a smaller part of the N-terminal domain (amino acids 360-528) was sufficient for the constitutively active AR to induce transcription of the same (GRE)2tkCAT reporter in HeLa cells. This demonstrates the capacity of the AR to use different regions in the N-terminal domain as transcription activation units (TAUs). To obtain additional information of AR N-terminal TAUs, the GAL4 DNA binding domain was linked to either the entire or parts of the AR N-terminal domain and cotransfected with the (UAS)2tkCAT reporter in HeLa cells. The results confirmed that the first 485 amino acid residues accommodate a transcription activation function. When the chimeric AR-GAL4 constructs were tested on a different reporter ((UAS)5E1bCAT), a small shift in position of the TAU, responsible for full transcription activation, was observed. The data presented show that the size and location of the active TAU in the human AR is variable, being dependent on the promoter context and the presence or absence of the ligand binding domain. To locate in detail the regions in the human androgen receptor (AR) involved in transcription activation, a series of N-terminal deletions was introduced in the wild type AR and in a constitutively active AR. The different constructs were tested for their capacity to activate transcription. Almost the entire N-terminal domain (residues 1-485) was necessary for full wild type AR activity when cotransfected with the (GRE)2tkCAT reporter in HeLa cells. In contrast, a smaller part of the N-terminal domain (amino acids 360-528) was sufficient for the constitutively active AR to induce transcription of the same (GRE)2tkCAT reporter in HeLa cells. This demonstrates the capacity of the AR to use different regions in the N-terminal domain as transcription activation units (TAUs). To obtain additional information of AR N-terminal TAUs, the GAL4 DNA binding domain was linked to either the entire or parts of the AR N-terminal domain and cotransfected with the (UAS)2tkCAT reporter in HeLa cells. The results confirmed that the first 485 amino acid residues accommodate a transcription activation function. When the chimeric AR-GAL4 constructs were tested on a different reporter ((UAS)5E1bCAT), a small shift in position of the TAU, responsible for full transcription activation, was observed. The data presented show that the size and location of the active TAU in the human AR is variable, being dependent on the promoter context and the presence or absence of the ligand binding domain. Steroid hormone receptors constitute one of the best available model systems for studying regulation of gene transcription. Based upon phylogenetic studies, the family of nuclear receptors can be grouped into three subfamilies: 1) steroid receptors to which also the androgen receptor (AR) 1The abbreviations used are: ARandrogen receptorHREhormone response elementGREglucocorticoid response elementtkthymidine kinaseMRmineralocorticoid receptorGRglucocorticoid receptorPRprogesterone receptorTAUtranscription activation unitDBDDNA binding domainCATchloramphenicol acetyltransferaseGDBDGAL4 DNA binding domainUASupstream-activating sequencesdamDNA adenine methylase. belongs, 2) thyroid hormone and retinoid receptors, and 3) orphan receptors that lack a well defined ligand(1Laudet V. Hänni C. Coll J. Catzeflis F. Stéhelin D. EMBO J. 1992; 11: 1003-1013Crossref PubMed Scopus (559) Google Scholar). Upon testosterone or dihydrotestosterone binding, the AR undergoes several sequential processes to interact with cognate DNA sequences. These DNA sequences (hormone response elements (HREs)) are commonly located in the regulating regions of the target genes. The binding of the AR to the HRE results in the formation of a stable preinitiation complex near the transcription start site, which allows efficient transcription initiation by RNA polymerase II. The mechanism by which steroid receptors stabilize or interact with the preinitiation complex is poorly understood. There is experimental evidence for a direct interaction of receptor with the general transcription factor IIB(2Ing N.H. Beekman J.M. Tsai S.Y. Tsai M.-J. O'Malley B.W. J. Biol. Chem. 1992; 267: 17617-17623Abstract Full Text PDF PubMed Google Scholar, 3Baniahmad A. Ha I. Reinberg D. Tsai S. Tsai M.-J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8832-8836Crossref PubMed Scopus (300) Google Scholar). Furthermore, steroid receptors could indirectly associate with the preinitiation complex via so called bridging factors (co-activators) or could make promoters accessible for other transcription activators by nucleosome displacement (reviewed in (4Archer T.K. Lefebvre P. Wolford R.G. Hager G.L. Science. 1992; 255: 1573-1576Crossref PubMed Scopus (347) Google Scholar, 5Gill G. Tjian R. Curr. Opin. Genet. Dev. 1992; 2: 236-242Crossref PubMed Scopus (104) Google Scholar, 6Truss M. Chalepakis G. Beato M. J. Steroid Biochem. Mol. Biol. 1992; 43: 365-378Crossref PubMed Scopus (38) Google Scholar, 7Adams C.C. Workman J.L. Cell. 1993; 72: 305-308Abstract Full Text PDF PubMed Scopus (139) Google Scholar, 8Wolffe A.P. Cell. 1994; 77: 13-16Abstract Full Text PDF PubMed Scopus (225) Google Scholar)). androgen receptor hormone response element glucocorticoid response element thymidine kinase mineralocorticoid receptor glucocorticoid receptor progesterone receptor transcription activation unit DNA binding domain chloramphenicol acetyltransferase GAL4 DNA binding domain upstream-activating sequences DNA adenine methylase. All nuclear receptors are composed of at least four functional domains: the N-terminal domain, DNA binding domain (DBD), hinge region, and the ligand binding domain(9Green S. Chambon P. Trends Genet. 1988; 4: 309-314Abstract Full Text PDF PubMed Scopus (826) Google Scholar, 10Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6218) Google Scholar, 11Truss M. Beato M. Endocr. Rev. 1993; 14: 459-479Crossref PubMed Scopus (588) Google Scholar). The DNA binding domain, composed of two “zinc-finger” structures, is, like the C-terminal ligand binding domain, highly conserved among the steroid receptors(1Laudet V. Hänni C. Coll J. Catzeflis F. Stéhelin D. EMBO J. 1992; 11: 1003-1013Crossref PubMed Scopus (559) Google Scholar). Interestingly, although the N-terminal domain of all steroid receptors harbors a transcription activation function, its length and amino acid residue constitution is unique for each receptor(12Kumar V. Green S. Stack G. Berry M. Jin J.-R. Chambon P. Cell. 1987; 51: 941-951Abstract Full Text PDF PubMed Scopus (1044) Google Scholar, 13Giguere V. Hollenberg S.M. Rosenfeld M.G. Evans R.M. Cell. 1986; 46: 645-652Abstract Full Text PDF PubMed Scopus (666) Google Scholar, 14Dobson A.D.W. Conneely O.M. Beattie W. Maxwell B.L. Mak P. Tsai M.-J. Schrader W.T. O'Malley B.W. J. Biol. Chem. 1989; 264: 4207-4211Abstract Full Text PDF PubMed Google Scholar, 15Gronemeyer H. Turcotte B. Quirin-Stricker C. Bocquel M.T. Meyer M.E. Krozowski Z. Jeltsch J.M. Lerouge T. Garnier J.M. Chambon P. EMBO J. 1987; 6: 3985-3994Crossref PubMed Scopus (162) Google Scholar, 16Jenster G. van der Korput J.A.G.M. van Vroonhoven C. van der Kwast Th. H. Trapman J. Brinkmann A.O. Mol. Endocrinol. 1991; 5: 1396-1404Crossref PubMed Scopus (405) Google Scholar, 17Simental J.A. Sar M. Lane M.V. French F.S. Wilson E.M. J. Biol. Chem. 1991; 266: 510-518Abstract Full Text PDF PubMed Google Scholar, 18Rupprecht R. Arriza J.L. Spengler D. Reul J.M.H.M. Evans R.M. Holsboer F. Damm K. Mol. Endocrinol. 1993; 7: 597-603Crossref PubMed Scopus (120) Google Scholar). The sizes of the N-terminal domain of the androgen, glucocorticoid (GR), progesterone (PR), and mineralocorticoid receptors (MR) are approximately half of the total receptor size. Compared with other nuclear receptors, this is exceptionally large, and it coincides with the observation that the AR, GR, PR, and MR are the only nuclear receptors recognizing HREs that fit consensus glucocorticoid response elements (GREs)(1Laudet V. Hänni C. Coll J. Catzeflis F. Stéhelin D. EMBO J. 1992; 11: 1003-1013Crossref PubMed Scopus (559) Google Scholar, 19Forman B.M. Samuels H.H. Mol. Endocrinol. 1990; 4: 1293-1301Crossref PubMed Scopus (345) Google Scholar, 20Freedman L.P. Luisi B.F. J. Cell. Biochem. 1993; 51: 140-150Crossref PubMed Scopus (102) Google Scholar). The specific GRE binding is reflected by the presence of a glycine, serine, and valine residue in the so called P-box located in the first zinc finger of the DNA binding domain (21Danielsen M. Hinck L. Ringold G.M. Cell. 1989; 57: 1131-1138Abstract Full Text PDF PubMed Scopus (256) Google Scholar, 22Mader S. Kumar V. de Verneuil H. Chambon P. Nature. 1989; 338: 271-274Crossref PubMed Scopus (355) Google Scholar, 23Zilliacus J. Dahlman-Wright K. Wright A. Gustafsson J.-A. Carlstedt-Duke J. J. Biol. Chem. 1991; 266: 3101-3106Abstract Full Text PDF PubMed Google Scholar). Based on the P-box sequence, the AR, GR, PR, and MR are classified in the “GSckV group”(1Laudet V. Hänni C. Coll J. Catzeflis F. Stéhelin D. EMBO J. 1992; 11: 1003-1013Crossref PubMed Scopus (559) Google Scholar, 19Forman B.M. Samuels H.H. Mol. Endocrinol. 1990; 4: 1293-1301Crossref PubMed Scopus (345) Google Scholar). The residues in the P-box determine which type of HRE half-site (consensus GRE half-site, TGTTCT or estrogen response element half-site, TGACCT) the receptor recognizes (21Danielsen M. Hinck L. Ringold G.M. Cell. 1989; 57: 1131-1138Abstract Full Text PDF PubMed Scopus (256) Google Scholar, 22Mader S. Kumar V. de Verneuil H. Chambon P. Nature. 1989; 338: 271-274Crossref PubMed Scopus (355) Google Scholar, 23Zilliacus J. Dahlman-Wright K. Wright A. Gustafsson J.-A. Carlstedt-Duke J. J. Biol. Chem. 1991; 266: 3101-3106Abstract Full Text PDF PubMed Google Scholar, 24Umesono K. Evans R.M. Cell. 1989; 57: 1139-1146Abstract Full Text PDF PubMed Scopus (708) Google Scholar). Almost all members of the thyroid hormone, retinoid receptor, and orphan receptor subfamilies bind HREs containing estrogen response element-like half-sites and are classified in the “EGckA/EGckG/EGckS” P-box groups(1Laudet V. Hänni C. Coll J. Catzeflis F. Stéhelin D. EMBO J. 1992; 11: 1003-1013Crossref PubMed Scopus (559) Google Scholar, 19Forman B.M. Samuels H.H. Mol. Endocrinol. 1990; 4: 1293-1301Crossref PubMed Scopus (345) Google Scholar). In these subfamilies, different mechanisms have been described that enlarge diversity and could explain, at least partially, cell and promoter discrimination: receptor heterodimerization, variable spacing of the HRE half-sites, and direct repeat or inverted repeat orientation of the two HRE half-sites(25Zechel C. Shen X.-Q. Chambon P. Gronemeyer H. EMBO J. 1994; 13: 1414-1424Crossref PubMed Scopus (173) Google Scholar, 26Zechel C. Shen X.-Q. Chen J.-Y. Chen Z.-P. Chambon P. Gronemeyer H. EMBO J. 1994; 13: 1425-1433Crossref PubMed Scopus (230) Google Scholar, 27De Luca L.M. FASEB J. 1991; 5: 2924-2933Crossref PubMed Scopus (806) Google Scholar, 28Glass C.K. DiRenzo J. Kurokawa R. Han Z. DNA Cell Biol. 1991; 10: 623-638Crossref PubMed Scopus (89) Google Scholar, 29Green S. Nature. 1993; 361: 590-591Crossref PubMed Scopus (98) Google Scholar, 30Giguere V. Endocr. Rev. 1994; 15: 61-79Crossref PubMed Google Scholar). Since these variabilities have not been found for the “GSckV class” receptors, it seems reasonable to assume that the large and unique N-terminal domains of the AR, GR, PR, and MR are important in cell- and receptor-specific regulation of target genes through multiple N-terminal TAUs (also referred to as TAF, AF, or TAD). For some promoters, experimental evidence has been provided, showing that promoter context as well as receptor N-terminal domain could determine receptor specificity(31Adler A.J. Danielsen M. Robins D.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11660-11663Crossref PubMed Scopus (131) Google Scholar, 32Pearce D. Yamamoto K.R. Science. 1993; 259: 1161-1165Crossref PubMed Scopus (395) Google Scholar). Several different sequence motifs that characterize transcription activation units have been identified thus far, including acidic regions (acidic activation domains), proline-rich domains, and glutamine-rich domains (reviewed in (5Gill G. Tjian R. Curr. Opin. Genet. Dev. 1992; 2: 236-242Crossref PubMed Scopus (104) Google Scholar) and (33Mitchell P.J. Tjian R. Science. 1989; 245: 371-378Crossref PubMed Scopus (2165) Google Scholar) −35). Although the AR N-terminal domain does not possess a significant sequence identity with other known transcription factors, it is glutamine and proline rich (including homopolymeric Gln and Pro stretches) and has a relatively high number of acidic amino acids(36Faber P.W. Kuiper G.G.J.M. van Rooij H.C.J. van der Korput J.A.G.M. Brinkmann A.O. Trapman J. Mol. Cell. Endocrinol. 1989; 61: 257-262Crossref PubMed Scopus (105) Google Scholar). To investigate the size and location of the TAU responsible for the transactivating capacity of the human AR in different situations, a series of N-terminal deletions was introduced in the wild type AR and in a constitutively active AR. These AR mutants were tested for their ability to activate transcription of an androgen-responsive reporter gene. In addition, chimeric AR-GAL4 DNA binding domain constructs were generated to obtain additional evidence for the localization of TAU regions. COS-1 and HeLa cells were maintained in Eagle's minimum essential medium supplemented with 5% fetal calf serum, nonessential amino acids, and antibiotics. The cells were cultured in 6-cm Ø dishes (3 × 105 cells/dish) and transfected using the calcium-phosphate precipitation method (2.5 μg of AR plasmid, 2.5 μg of reporter plasmid, and 5 μg of pTZ19 carrier plasmid/dish)(37Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 1745-1752Google Scholar). The cells were plated and transfected in medium containing 5% dextran/charcoal-treated fetal calf serum. 24 h prior to cell lysis, cells were incubated with 1 nM R1881 (17α-methyl-trienolone) (DuPont NEN). The CAT assays were performed essentially as previously described(38Seed B. Sheen J.-Y. Gene (Amst.). 1988; 67: 271-277Crossref PubMed Scopus (830) Google Scholar). Transfections of complete series of AR mutants were performed five times in duplicate, using at least two independent plasmid extracts. In each experiment, CAT activities were corrected for the reporter background, and the percentage relative to that of the total number of counts in the assay was determined for each mutant. The means (±S.E.) were calculated, and the starting mutants (AR0, AR5, or AR4G) were set to 100%. A human AR cDNA expression vector (pAR0) was constructed using the SV40 early promoter and the rabbit β-globin polyadenylation signal(39Brinkmann A.O. Faber P.W. van Rooij H.C.J. Kuiper G.G.J.M. Ris C. Klaassen P. van der Korput J.A.G.M. Voorhorst M.M. van Laar J.H. Mulder E. Trapman J. J. Steroid Biochem. 1989; 34: 307-310Crossref PubMed Scopus (272) Google Scholar). The AR cDNA encodes 910 amino acids, of which the polymorphic glutamine stretch contains 20 Gln residues and the glycine stretch consists of 16 Gly residues. Construction of AR mutants pAR22 and pAR61 has been described(16Jenster G. van der Korput J.A.G.M. van Vroonhoven C. van der Kwast Th. H. Trapman J. Brinkmann A.O. Mol. Endocrinol. 1991; 5: 1396-1404Crossref PubMed Scopus (405) Google Scholar). The AR mutants pAR3, pAR9 (deletion codons 46-101), pAR10 (deletion codons 100-142), pAR11 (deletion codons 139-188), pAR19 (deletion codons 186-244 and an additional mutation in codon 249 resulting in Glu to Val substitution), pAR34 (deletion codons 626-654), pAR53 (deletion codons 550-593), and pAR62 (deletion codons 485-528) were constructed by site-directed mutagenesis using polymerase chain reaction DNA amplification techniques(40Higuchi R. Krummel B. Saiki R.K. Nucleic Acids Res. 1988; 16: 7351-7367Crossref PubMed Scopus (2067) Google Scholar). In the pAR3 plasmid, the unique XbaI, ClaI, and MluI restriction sites were inserted in between the second and third codon after the ATG, resulting in five additional codons coding for amino acids SRSIR. In the AR mutants pAR9, pAR10, pAR11, pAR19, pAR34, pAR53, and pAR62, a unique ClaI site was introduced in the same matching frame in all constructs (identical to the ClaI site present in pAR22 and pAR3). In this way, parts of the AR cDNA can be swapped easily between these mutants, resulting in all kinds of in-frame deletions or duplications. Introduction of the ClaI site (in the format A-TCG-AT∗) will always encode the Ser-Met or Ser-Ile amino acids. This motif was not always present on the borders of the deletion. Therefore, the mutants pAR9, pAR10, pAR34, and pAR53 contain additional modifications on the borders of the deletion (for details, see primers). In several AR mutant constructs (pAR3, pAR9, pAR10, pAR34, and pAR53), it was chosen to introduce a DNA adenine methylase (dam) methylation site (GATC), making ClaI unable to digest when the plasmid was multiplied in the dam-positive Escherichia coli strain DH5α. This inability to digest was easily abolished when the plasmid was multiplied in the dam-negative E. coli strain GM121. The following primers introducing the insertion or deletion were used: AR3, 5′CAAGCTCAAGGATGGAATCTAGATCGATACGCGTGCAGTTAGGGCTG-3′; AR9, 5′-AGGCACCCAGAGGCCGCATCGATCACAGGCTACCTGGTC-3′; AR10, 5′-CCAAGCCCATCGTAGATCGATGCCGCAGCAGCTGCCA-3′; AR11, 5′-GAGCCGCCGTGGCCGCATCGATGCAACTCCTTCAGC-3′; AR19, 5′-ACATCCTGAGCGAGGCATCGATGGGCCTGGGTGTGG-3′; AR34, 5′-GCTGAAGAAACTTGGATCGATTGAAGGCTATGAATG-3′; AR53, 5′-CTTTCCACCCCAGAGATCGATTGATAAATTCGGA-3′; and AR62, 5′-GCTGGCGGGCCAGGAATCGATGCGTTTGGAGACTG-3′. All of these AR mutants were sequenced to verify the correct reading frame before they were used as starting constructs for the following AR mutants. pAR110 was constructed by removing the 2.8-kilobase ClaI-BamHI fragment, containing most of the AR cDNA, from pAR3 and insertion of the 2.5-kilobase ClaI-BamHI fragment from pAR9 into pAR3. In the same way, pAR120 (combination of pAR3 and pAR10), pAR121 (combination of pAR3 and pAR11), pAR123 (combination of pAR3 and pAR22), pAR104 (combination of pAR3 and pAR62), pAR60 (combination of pAR22 and pAR62), and pAR55 (combination of pAR9 and pAR19) were constructed. pAR113 was constructed by removal of the internal fragment of pAR62 digested with RsrII and ClaI. Ligation of the blunt-ended plasmid resulted in an in-frame deletion. pAR130 and pAR131 were constructed by combining the deletions of pAR110 and pAR62, and pAR110 and pAR113, respectively. pAR5 was constructed by ligation of a XbaI linker (Promega) that contains an in-frame stop codon into the blunt-ended ClaI site of pAR34. pAR124, pAR127, pAR106, pAR99, pAR126, pAR98, pAR100, pAR115, pAR117, and pAR132 were constructed by introduction of the deletion present in pAR120, pAR121, pAR123, pAR62, pAR113, pAR60, pAR55, pAR22, pAR61, and pAR104, respectively, into pAR5. pAR105 was constructed by combining the pAR3 and pAR100 mutants as described above. pAR128 was constructed by combining the deletions of pAR106 and pAR99. pAR0G was constructed by insertion of the ClaI-digested polymerase chain reaction fragment encoding the GAL4 DNA binding domain (GDBD, amino acids 1-147) into the ClaI-digested pAR53 vector. The orientation and sequence of the polymerase chain reaction insert was checked. The following primers were used on the pSG424 plasmid (41Sadowski I. Ptashne M. Nucleic Acids Res. 1989; 17: 7539Crossref PubMed Scopus (467) Google Scholar) template to obtain the correct GAL4 DBD fragment (GDBD-A (5′-primer), 5′-CAAGCCTCCTGATCGATGAAGCTACTG-3′ and GDBD-B (3′-primer), 5′-CCCGGGAATTCCATCGATACAGTCAAC-3′). pAR4G was constructed by ligation of a XbaI linker that contains an in-frame stop codon into the blunt-ended ClaI site of pAR0G (multiplied in E. coli DH5α). Please notice that pAR0G contains two ClaI sites but that only the ClaI site 5′ of the GDBD insert is dam methylated. pAR94G, pAR107G, pAR105G, pAR106G, pAR99G, pAR113G, pAR98G, pAR100G, pAR115G, and pAR117G were constructed by introduction of the deletion present in pAR124, pAR127, pAR105, pAR106, pAR99, pAR126, pAR98, pAR100, pAR115, or pAR117, respectively, into pAR4G. pAR96G, pAR91G, pAR85G, and pAR84G were constructed by combining the deletions of pAR94G and pAR99G, pAR107G and pAR99G, pAR94G and pAR113G, and pAR107G and pAR113G, respectively. All mutants were expressed in COS-1 cells, and the AR proteins were analyzed by Western blotting(16Jenster G. van der Korput J.A.G.M. van Vroonhoven C. van der Kwast Th. H. Trapman J. Brinkmann A.O. Mol. Endocrinol. 1991; 5: 1396-1404Crossref PubMed Scopus (405) Google Scholar). Using the antibodies SP197, SP061, SP066, F52.24.4, F39.4.1(42Kuiper G.G.J.M. de Ruiter P.E. Trapman J. Boersma W.J.A. Grootegoed J.A. Brinkmann A.O. Biochem. J. 1993; 291: 95-101Crossref PubMed Scopus (53) Google Scholar, 43Zegers N.D. Claassen E. Neelen C. Mulder E. van Laar J.H. Voorhorst M.M. Berrevoets C.A. Brinkmann A.O. van der Kwast Th. H. de Winter J.A.R. Trapman J. Boersma W.J.A. Biochim. Biophys. Acta. 1991; 1073: 23-32Crossref PubMed Scopus (94) Google Scholar, 44van Laar J.H. Voorhorst-Ogink M.M. Zegers N.D. Boersma W.J.A. Claassen E. van der Korput J.A.G.M. Ruizeveld de Winter J.A. van der Kwast Th. H. Mulder E. Trapman J. Brinkmann A.O. Mol. Cell. Endocrinol. 1989; 67: 29-38Crossref PubMed Scopus (54) Google Scholar, 45Veldscholte J. Berrevoets C.A. Zegers N.D. van der Kwast Th. H. Grootegoed J.A. Mulder E. Biochemistry. 1992; 31: 7422-7430Crossref PubMed Scopus (113) Google Scholar), and 2GV3/3GV2 (46White J. Brou C. Wu J. Lutz Y. Moncollin V. Chambon P. EMBO J. 1992; 11: 2229-2240Crossref PubMed Scopus (95) Google Scholar) all mutants except AR106 could be visualized. The expression levels of the various mutants were comparable. AR106 could only be detected using the F52.24.4 antibody. This monoclonal antibody is not specific enough to use directly for development of Western blots loaded with whole cell lysates, since it recognizes many zinc finger-containing proteins. We were unable to detect AR106 after immunoprecipitation, most likely because AR106 (calculated molecular mass is approximately 28 kDa) comigrated with the light chain of the F52.24.4 antibody. Additionally, the key mutants pAR0, pAR5, pAR0G, and pAR4G were checked for nuclear localization(47Jenster G. Trapman J. Brinkmann A.O. Biochem. J. 1993; 293: 761-768Crossref PubMed Scopus (210) Google Scholar). The pG29GtkCAT ((GRE)2tkCAT) reporter plasmid (48Schüle R. Muller M. Kaltschmidt C. Renkawitz R. Science. 1988; 242: 1418-1420Crossref PubMed Scopus (384) Google Scholar) contains two progesterone/glucocorticoid-responsive elements in front of the thymidine kinase (tk) promoter linked to the CAT gene. The (UAS)2tkCAT reporter (kindly provided by Dr. R. Renkawitz and Dr. M. Muller) is in principle the same except that the two progesterone/glucocorticoid-responsive elements are exchanged by two upstream-activating sequences (UAS) to which the GAL4 DNA binding domain can bind(49Baniahmad C. Muller M. Altschmied J. Renkawitz R. J. Mol. Biol. 1991; 222: 155-165Crossref PubMed Scopus (24) Google Scholar). The G5E1bCAT reporter contains 5 UASs in front of the E1b TATA box linked to the CAT gene(50Lillie J.W. Green M.R. Nature. 1989; 338: 39-44Crossref PubMed Scopus (471) Google Scholar). A series of AR N-terminal deletion mutants was tested for transcription activity in HeLa cells using the (GRE)2tkCAT reporter (Fig. 1) (pG29GtkCAT)(48Schüle R. Muller M. Kaltschmidt C. Renkawitz R. Science. 1988; 242: 1418-1420Crossref PubMed Scopus (384) Google Scholar). Cotransfection of the wild type AR expression plasmid (pAR0) together with the reporter construct in the presence of 1 nM R1881 (a non-metabolizable androgen), resulted in a 20-40-fold induction of CAT expression as compared with the CAT activity of the unliganded AR (data not shown). N-terminal deletion of the first 100 or 141 amino acid residues (AR110 and AR120, respectively) resulted in a reduction to approximately 60% activity as compared with wild type AR (Fig. 1). Further deletion to amino acids 188 (AR121), 360 (AR123), or 528 (AR104) gradually reduced the transcription activation capacity of the respective AR mutants. These results suggest that almost the entire N-terminal domain is involved in transcription activation because no sharp N-terminal border of a TAU could be marked. To locate the C-terminal boundary of the TAU responsible for the wild type AR-transactivating capacity, AR62 (Δ485-528), AR113 (Δ370-528), and AR60 (Δ244-528) were generated and tested for activity. Deletion of the last 42 residues of the AR N terminus (AR62) had no effect on transcription activity, whereas deletion of 157 residues (AR113) reduced transcription activity to approximately 70%. AR60, which lacks the second half of the N-terminal domain, is hardly capable of activating transcription of the (GRE)2tkCAT reporter. Importantly, the inability of AR60 to activate transcription could not be attributed to a smaller deletion. AR22 (Δ244-360), AR61 (Δ360-494), and AR62 (Δ485-528) contain smaller deletions that together cover the large region deleted in AR60. All three AR mutants, however, were quite capable of inducing transcription. This indicates that the TAU function cannot be attributed to a single small structural element and suggests the presence of several different TAU elements that have the capacity to compensate for loss of transcription activity due to deletion of one of them. Deletion of the region between amino acids 46-244 in the first half of the N-terminal domain (AR55) also resulted in a major reduction of transactivating capacity. This mutant showed that although the deletion of amino acids 244-528 (AR60) strongly affected transcription activity, these sequences by themselves (AR55) are not sufficient for transcription activation. To locate the minimal part of the N-terminal domain that still retained most of the transactivation capacity, AR130 and AR131 were constructed. Although both mutants were quite capable of inducing transcription (approximately 65% compared with the wild type AR0), the only AR mutant retaining full transcription activity was AR62, which again indicates that almost the entire AR N terminus is necessary for wild type receptor activity (residues 1-485, designated TAU-1). To obtain information on the size and location of the TAU responsible for the transactivating capacity of a constitutively active AR, a construct was generated that lacks the ligand binding domain (AR5). Compared with the wild type AR, the transcription activation capacity of AR5 was approximately 75%. R1881 had no effect on the transcription induction of the constitutively active receptor. The same series of deletions as analyzed in AR0 derivatives was introduced in the constitutively active AR5. The capacity of these constructs to activate transcription was, like the wild type AR deletion series, tested in HeLa cells using the same (GRE)2tkCAT reporter. Surprisingly, in contrast to the results found in the AR0 deletion analysis, truncation of the first 360 amino acids in the constitutively active AR (AR106) did not result in a decrease in the transactivating capacity, indicating a TAU in the 360-528 region (Fig. 2). All other deletion mutants confirm the presence of a TAU in this area (designated TAU-5). Deletions of part of the 360-528 domain resulted in complete loss of or decreased transactivating capacity (AR99, AR126, AR98, and AR117). In contrast, in the mutants AR100, AR115, AR124, AR127, and AR105, in which the 360-528 region is present, complete (or almost complete) transactivating capacity was observed. So, the smallest N-terminal region responsible for full AR5 transcription activity is located between residues 360 and 528. To obtain additional evidence for the presence of a TAU in the AR N-terminal domain, a construct was generated containing the first 550 amino acids of the AR, linked to the GAL4 DNA binding domain (AR4G). In addition, a series of constructs was generated encoding several different parts of the AR N-terminal domain linked to the GAL4 DBD. Chimeric proteins were tested for transactivating capacity in HeLa cells using the (UAS)2tkCAT reporter (Fig. 3, filled bars). The (UAS)2tkCAT reporter is comparable with the (GRE)2tkCAT reporter construct used for the wild type and constitutively active AR mutants, except that the two GRE sequences have been replaced by UASs to which the GAL4 DNA binding domain can bind(49Baniahmad C. Muller M. Altschmied J. Renkawitz R. J. Mol. Biol. 1991; 222: 155-165Crossref PubMed Scopus (24) Google Scholar). It could be expected that the TAU, responsible for the transactivating capacity of the constitutively active AR4G, would be located in the same region (amino acids 360-528) as TAU-5, which is responsible for the constitutive AR5-transactivating capacity. However, enlargement of the N-terminal truncation (AR94G, AR107G, AR105G, AR106G) resulted in a gradual decrease in transcription activity when tested on the (UAS)2tkCAT reporter (Fig. 3, filled bars). The region between amino acids 370 and 550 is still essential for transcription activity of AR4G since the transactivating capacity of AR113G (which lacks amino acids 370-550) was strongly reduced. The TAU responsible for full AR4G activity, when tested on the (UAS)2tkCAT reporter, is located between amino acids 188 and 485 (AR91G) (Fig. 3). The same series of chimeric proteins was tested on the (UAS)5E1bCAT construct (Fig. 3, empty bars). This reporter contains five UASs upstream of the E1bTATA box and represents a different promoter environment(50Lillie J.W. Green M.R. Nature. 1989; 338: 39-44Crossref PubMed Scopus (471) Google Scholar). The transcription activation capacities of the chimeric proteins were different when tested on the (UAS)5E1bCAT reporter. A large reduction in transactivating capacity was observed between mutants AR94G (Δ1-142) and AR107G (Δ1-188), which shows that the region between amino acids 142-188 is essential for the chimeric proteins to be able to activate transcription of the (UAS)5E1bCAT reporter. In contrast, the capacities of AR94G and AR107G to activate transcription of the (UAS)2tkCAT reporter were not significantly different. This promoter specificity, determined by AR amino acids 142-188, is also obvious for mutants AR100G and AR91G (Fig. 3). On the other hand, the region between amino acids 360 and 550 is more important for the chimeric proteins to be able to activate transcription of the (UAS)2tkCAT construct. AR106G, which only contains this last part of the AR N-terminal domain, was incapable of inducing transcription when cotransfected with the (UAS)5E1bCAT construct but retained approximately 35% activity when tested on the (UAS)2tkCAT reporter. When tested on the (UAS)5E1bCAT reporter, the minimal part of the AR N-terminal domain still capable of inducing full transcription activity is located between residues 142 and 485 (AR96G). To dissect the transcription activation properties of the human AR, a series of AR deletion mutants was analyzed to characterize and locate N-terminal regions essential for transcription activity. Deletion mapping of the wild type AR revealed that for full receptor activity, almost the entire N-terminal domain is necessary (TAU-1, residues 1-485) (Fig. 4). Any deletion, except for the deletion of the last 42 residues in the AR N terminus, affected the capacity to induce transcription when analyzed in HeLa cells using the (GRE)2tkCAT reporter. The minimal region (designated core region) that still retained over 50% transcription activity compared with AR0, was located between residues 101 and 370 (Fig. 4). Interestingly, these results are clearly different from the data obtained in the deletion mapping studies of the constitutively active AR5. The same series of deletions was introduced in AR5, and the respective mutants were analyzed under identical experimental conditions (HeLa cells using the (GRE)2tkCAT reporter). These studies revealed that a region between amino acids 360 and 528 (designated TAU-5) was sufficient for full constitutive transcription activity (Fig. 4). This demonstrates the capability of the AR to use different N-terminal regions for transcription activation. Furthermore, these observations show a determinant role for the ligand binding domain in TAU functioning. Deletion of the ligand binding domain resulted in loss of TAU-1 activity and induced the use of TAU-5. These findings might indicate a functional interaction between the ligand binding domain and the AR N-terminal domain. Such an interaction has already been suggested by McPhaul and co-workers(51McPhaul M.J. Marcelli M. Tilley W.D. Griffin J.E. Isidro-Gutierrez R.F. Wilson J.D. J. Clin. Invest. 1991; 87: 1413-1421Crossref PubMed Scopus (106) Google Scholar). They examined a mutant AR, which contained two structural alterations: a shortened N-terminal glutamine stretch and a tyrosine to cysteine substitution in the ligand binding domain(51McPhaul M.J. Marcelli M. Tilley W.D. Griffin J.E. Isidro-Gutierrez R.F. Wilson J.D. J. Clin. Invest. 1991; 87: 1413-1421Crossref PubMed Scopus (106) Google Scholar). Interestingly, the ability of the AR to activate transcription was strongly diminished only when both alterations were present, indicating a potential interaction between the two different domains. The observation that deletion of the ligand binding domain induces the use of TAU-5, however, does not permit one to draw conclusions with respect to its functional in vivo existence, since so far, constitutively active ARs have not been identified in intact in vivo systems. Although TAU-1 and TAU-5 overlap, the core of the two TAUs (responsible for over 50% of the activity) are separate N-terminal regions with individual characteristics. TAU-1 contains a relatively high number of acidic amino acids, three glutamine repeats of which one is polymorphic, and potential phosphorylation sites(36Faber P.W. Kuiper G.G.J.M. van Rooij H.C.J. van der Korput J.A.G.M. Brinkmann A.O. Trapman J. Mol. Cell. Endocrinol. 1989; 61: 257-262Crossref PubMed Scopus (105) Google Scholar, 42Kuiper G.G.J.M. de Ruiter P.E. Trapman J. Boersma W.J.A. Grootegoed J.A. Brinkmann A.O. Biochem. J. 1993; 291: 95-101Crossref PubMed Scopus (53) Google Scholar). The TAU-5 core is not acidic and harbors three different amino acid stretches: 1) a proline stretch (residues 371-378), 2) an alanine stretch (residues 397-401), and 3) a glycine stretch (residues 448-463)(36Faber P.W. Kuiper G.G.J.M. van Rooij H.C.J. van der Korput J.A.G.M. Brinkmann A.O. Trapman J. Mol. Cell. Endocrinol. 1989; 61: 257-262Crossref PubMed Scopus (105) Google Scholar). The role of the different amino acid stretches in the TAU-5 region is not known. Lengthening of the Gln repeat to more than 40 residues is associated with Kennedy's disease, an X-linked neurodegenerative disorder, characterized by a slowly progressing muscle weakness(52La Spada A.R. Wilson E.M. Lubahn D.B. Harding A.E. Fischbeck K.H. Nature. 1991; 352: 77-79Crossref PubMed Scopus (2318) Google Scholar). Mhatre and co-workers (53Mhatre A.N. Trifiro M.A. Kaufman M. Kazemi-Esfarjani P. Figlewicz D. Rouleau G. Pinsky L. Nature Genet. 1993; 5: 184-188Crossref PubMed Scopus (316) Google Scholar) have shown that the extension of the Gln repeat to 40 or 50 residues resulted in a decreased AR capacity to activate transcription. The capability of the AR to use different and unique regions of its N-terminal domain as TAUs introduces the possibility that different TAUs are responsible for the regulation of different genes, resulting in cell-specific and AR-specific gene expression. Evidence for the use of different N-terminal regions to activate different kinds of promoters was provided by the analysis of a series of AR-GAL4 chimeric constructs. These proteins contained either the entire or part of the AR N-terminal domain linked to the GAL4 DNA binding domain. When the transactivating capacity was tested on two different promoters, a shift in position of the TAU responsible for full activity was observed. The location of the TAU was more C-terminal when tested on the (UAS)2tkCAT construct compared with the (UAS)5E1bCAT reporter (Fig. 4). It is unlikely that these two situations represent the use of two different and separate TAUs but rather indicate a small difference in the location of essential sequences. Since the AR4G and AR5 constructs only differ in their DBD, it might be expected that the respective TAUs would be located in the same part of the N-terminal domain. When AR4G derivatives were tested on the (UAS)2tkCAT reporter and the AR5 derivatives on the comparable (GRE)2tkCAT reporter, the region between amino acids 360-528 was essential for the transactivating capacity of both AR4G and AR5. However, for full transactivating capacity, the AR4G needs a larger region (amino acids 188-485), which might indicate that the replacement of the DBD influenced the size of the N-terminal region used as the TAU. In contrast to the DNA- and steroid binding domain, it is clear that the large and unique TAU-1 responsible for wild type AR transactivating capacity is not a sharply bordered functional domain. TAUs have been located and characterized in the N-terminal domains of the GR and PR. The N-terminal TAU of the human GR has been delineated to the central part of the GR N-terminal domain (amino acids 77-262). In contrast to our observations, the core unit of this TAU consists of only 40-60 amino acids(54Dahlman-Wright K. Almlöf T. McEwan I.J. Gustafsson J.-A. Wright A.P.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1619-1623Crossref PubMed Scopus (84) Google Scholar), and the same region is responsible for the wild type GR and the constitutively active GR functioning(55Hollenberg S.M. Evans R.M. Cell. 1988; 55: 899-906Abstract Full Text PDF PubMed Scopus (538) Google Scholar). The TAU, responsible for the transactivating capacity of the human PR B form, is located in the last 90 amino acids of the N-terminal domain, and its activity can be modulated by the first N-terminal 164 amino acids(56Meyer M.-E. Quirin-Stricker C. Lerouge T. Bocquel M.-T. Gronemeyer H. J. Biol. Chem. 1992; 267: 10882-10887Abstract Full Text PDF PubMed Google Scholar). These studies established that the location of the TAU in the various steroid receptors differs and that there is almost no evidence for sequence or structural homology. The sequences characterizing TAUs might represent interfaces that function by direct or indirect binding to general and/or specific transcription factors(2Ing N.H. Beekman J.M. Tsai S.Y. Tsai M.-J. O'Malley B.W. J. Biol. Chem. 1992; 267: 17617-17623Abstract Full Text PDF PubMed Google Scholar, 5Gill G. Tjian R. Curr. Opin. Genet. Dev. 1992; 2: 236-242Crossref PubMed Scopus (104) Google Scholar, 33Mitchell P.J. Tjian R. Science. 1989; 245: 371-378Crossref PubMed Scopus (2165) Google Scholar, 34Ptashne M. Nature. 1988; 335: 683-689Crossref PubMed Scopus (1159) Google Scholar, 35Tjian R. Maniatis T. Cell. 1994; 77: 5-8Abstract Full Text PDF PubMed Scopus (951) Google Scholar). However, the mechanism by which TAUs can regulate gene transcription is still largely unknown. We thank R. Renkawitz and M. Muller for the pG29GtkCAT and UAS-29-UAStkCAT constructs, P. Chambon for the 2GV3 and 3GV2 antibodies, J. A. Grootegoed for helpful discussions, and M. Tascilar and G. Meijer for technical assistance." @default.
• W2106571316 created "2016-06-24" @default.
• W2106571316 creator A5026981971 @default.
• W2106571316 creator A5034635531 @default.
• W2106571316 creator A5052912559 @default.
• W2106571316 creator A5056314091 @default.
• W2106571316 date "1995-03-01" @default.
• W2106571316 modified "2023-10-10" @default.
• W2106571316 title "Identification of Two Transcription Activation Units in the N-terminal Domain of the Human Androgen Receptor" @default.
• W2106571316 cites W1486980795 @default.
• W2106571316 cites W1495912963 @default.
• W2106571316 cites W1545280767 @default.
• W2106571316 cites W1572006943 @default.
• W2106571316 cites W1590197184 @default.
• W2106571316 cites W1595664534 @default.
• W2106571316 cites W1766657157 @default.
• W2106571316 cites W1843244908 @default.
• W2106571316 cites W1859376017 @default.
• W2106571316 cites W1939119178 @default.
• W2106571316 cites W1966068861 @default.
• W2106571316 cites W1973912514 @default.
• W2106571316 cites W1974015011 @default.
• W2106571316 cites W1974998400 @default.
• W2106571316 cites W1981765089 @default.
• W2106571316 cites W1989729537 @default.
• W2106571316 cites W1996554622 @default.
• W2106571316 cites W1997514802 @default.
• W2106571316 cites W1998108444 @default.
• W2106571316 cites W2000731870 @default.
• W2106571316 cites W2001034975 @default.
• W2106571316 cites W2002791165 @default.
• W2106571316 cites W2003002984 @default.
• W2106571316 cites W2004895743 @default.
• W2106571316 cites W2005914718 @default.
• W2106571316 cites W2008494281 @default.
• W2106571316 cites W2011158289 @default.
• W2106571316 cites W2022551939 @default.
• W2106571316 cites W2033501768 @default.
• W2106571316 cites W2033777731 @default.
• W2106571316 cites W2033900037 @default.
• W2106571316 cites W2037365073 @default.
• W2106571316 cites W2043888927 @default.
• W2106571316 cites W2053131171 @default.
• W2106571316 cites W2063110155 @default.
• W2106571316 cites W2066492784 @default.
• W2106571316 cites W2068577315 @default.
• W2106571316 cites W2068609431 @default.
• W2106571316 cites W2075263567 @default.
• W2106571316 cites W2075496313 @default.
• W2106571316 cites W2075788654 @default.
• W2106571316 cites W2082181748 @default.
• W2106571316 cites W2085124463 @default.
• W2106571316 cites W2093897768 @default.
• W2106571316 cites W2097852478 @default.
• W2106571316 cites W2113333419 @default.
• W2106571316 cites W2129972061 @default.
• W2106571316 cites W2162702340 @default.
• W2106571316 cites W2179648988 @default.
• W2106571316 cites W2292736786 @default.
• W2106571316 cites W2410727204 @default.
• W2106571316 cites W90893581 @default.
• W2106571316 doi "https://doi.org/10.1074/jbc.270.13.7341" @default.
• W2106571316 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7706276" @default.
• W2106571316 hasPublicationYear "1995" @default.
• W2106571316 type Work @default.
• W2106571316 sameAs 2106571316 @default.
• W2106571316 citedByCount "385" @default.
• W2106571316 countsByYear W21065713162012 @default.
• W2106571316 countsByYear W21065713162013 @default.
• W2106571316 countsByYear W21065713162014 @default.
• W2106571316 countsByYear W21065713162015 @default.
• W2106571316 countsByYear W21065713162016 @default.
• W2106571316 countsByYear W21065713162017 @default.
• W2106571316 countsByYear W21065713162018 @default.
• W2106571316 countsByYear W21065713162019 @default.
• W2106571316 countsByYear W21065713162020 @default.
• W2106571316 countsByYear W21065713162021 @default.
• W2106571316 countsByYear W21065713162022 @default.
• W2106571316 countsByYear W21065713162023 @default.
• W2106571316 crossrefType "journal-article" @default.
• W2106571316 hasAuthorship W2106571316A5026981971 @default.
• W2106571316 hasAuthorship W2106571316A5034635531 @default.
• W2106571316 hasAuthorship W2106571316A5052912559 @default.
• W2106571316 hasAuthorship W2106571316A5056314091 @default.
• W2106571316 hasBestOaLocation W21065713161 @default.
• W2106571316 hasConcept C104317684 @default.
• W2106571316 hasConcept C116834253 @default.
• W2106571316 hasConcept C121608353 @default.
• W2106571316 hasConcept C134306372 @default.
• W2106571316 hasConcept C138885662 @default.
• W2106571316 hasConcept C153911025 @default.
• W2106571316 hasConcept C179926584 @default.
• W2106571316 hasConcept C185592680 @default.
• W2106571316 hasConcept C2779664074 @default.
• W2106571316 hasConcept C2780192828 @default.
• W2106571316 hasConcept C33923547 @default.
• W2106571316 hasConcept C36503486 @default.
• W2106571316 hasConcept C41008148 @default.

• next