Patent Application: US-23241199-A

Abstract:
this invention provides a purified heterodimer comprising an rxr and a hormone receptor . the invention also provides a method of screening ligands for their effect on the activity of an rxr - containing hormone receptor heterodimer comprising combining the heterodimer with the ligand and determining the effect on activity . also provided is a method of amplifying the activity of a hormone receptor comprising forming a heterodimer with another hormone receptor .

Description:
the present invention is based on the core discovery that an rxr can form a heterodimer with other hormone receptors to increase the activity of the receptors . this increase can be in either the hormone receptors &# 39 ; activity or rxr &# 39 ; s activity . since rxrα and β are very closely related , rxrβhas a similar activity to rxrα . methods employing rxrβutilize the same methods , conditions etc . as set forth hereinbelow for rxrα . by “ activity ” is meant any activity which is affected by the heterodimer formation . generally , this activity is activation or enhancement of transcription . generally , the ligand of one or both hormone receptors of the heterodimer enhance the activity . by “ hormone receptor ” is meant a receptor of the steroid / thyroid hormone receptor superfamily which forms a heterodimer with an rxr . however , oligodimers are also covered herein . oligodimers can be tested by the methods set forth hereinbelow . moreover , any additional receptors not tested can be tested using the methods set forth herein . proteins having substantially the same sequence and activity of the receptors , such as “ rxr ”, are also included in the definition of hormone receptor . thus , minor substitutions , deletions and additions can readily be made and tested . moreover , any receptor consisting essentially of the amino acids of the hormone receptors are included in the definition . additionally , since heterodimer formation can be attributed to certain portions of the hormone receptors , molecules containing only those portions are also contemplated . also , since only certain regions of the receptor may be necessary for activity , i . e ., ligand or hormone binding region , heterodimers containing only these portions of the receptors are contemplated . the activities of the heterodimers can be applied to affect transcription in an in vivo system . thus , many therapeutic applications , including enhancement or inhibition of transcription , can readily be obtained . these methods can easily by adapted to use the heterodimers to screen further ligands for their effect on activity . in this way , more effective ligands can be determined . the well known methods used to screen ligands using a single receptor can readily be applied to screen using heterodimers . a key discovery set forth herein is that different receptors can form heterodimers with selective enhancement or reduction in activity . thereby specific genes can be regulated using the teachings herein . the following experimental procedures and results are set forth to exemplify and not limit the invention . the construction of reporter plasmids , tre - tk - cat and tre 2 - tk - cat has been described previously ( zhang et al ., 1991b ). the coding sequences of trα , trβ , rarβ , and rarγ were inserted into the multiple cloning sites of the eukaryotic expression vector pece or pbluescript ( stratagene ). the construction of these plasmids has been described ( graupner et al ., 1989 ; zhang et al ., 1991b ). rarα cdna was amplified from poly ( a ) rna prepared from the squamous cell carcinoma line , scc - 13 , by polymerase chain reaction ( pcr ). the pcr products were cloned into both pece and pbluescript . two primers ( seq . id no : 1 and seq . id no : 2 ) ( 5 ′- cgcagacatggacaccaaacat - 3 ′; 5 ′- cctctccaccggcatgtcctcg - 3 ′) were used to amplify the n - terminal half of rxrα cdna from scc - 13 by pcr technique . the smal - sall fragment from pcr product ( 530 bp ) containing the dna binding domain of the rxrα was used as a probe to isolate rxrα cdna by screening a λgt11 human placenta cdna library ( obtained from j . millán ; millán , 1986 ). several positive clones were obtained , including full length receptor and the truncated clones , rxrαm3 , rxrαm4 and rxrαm5 which were sequenced and show identical sequences as the wild type rxrα . the cdna clones were subsequently subcloned into the ecori site of pbluescript and pece . to obtain trα and rarγ deletion mutants , existing restriction enzyme sites on receptors were used to digest receptor cdnas . the resulting cdna fragments were purified and cloned into pbluescript . trαm1 and trαm2 were generated by digesting trα cdna with xhol ( 1530 ) and stul ( 964 ), respectively . rarγm1 , rarγm2 , and rarγm3 were generated by digesting rarγ cdna with pst 1 ( 1469 ), draiii ( 1066 ), and sac i ( 976 ), respectively ( numbers in brackets indicate the nucleotide position ). rxrα deletion mutants were obtained as following : rxrαm1 and rxrαm2 were generated by digesting rxrα cdna with stul ( 1463 ) and xmaiii ( 1231 ), respectively . rxrαm6 and rxrαm7 were generated by internal deletion using ncoi and ba1i , respectively . the construction of flag - containing receptors ( flag - rxrα , flag - trα , and flag - rarγ ) was described previously ( hermann et al ., 1991 ; zhang , et al ., 1991a ). briefly , they were constructed by ligating a double - stranded oligonucleotide containing an atg codon and a dna sequence encoding flag ( seq id no : 3 ) ( arg tyr lys asp asp asp asp lys ) ( hopp et al ., 1988 ) to the n - terminus of receptors . the fusion products were then cloned into pbluescript . cv - 1 cells were grown in dme medium supplemented with 10 % fetal calf serum ( fcs ). cells were plated at 1 . 0 × 10 5 per well in a 24 well plate 16 to 24 hours prior to transfection as described previously ( husmann et al ., 1991 ). a modified calcium phosphate precipitation procedure was used for transient transfection and is described elsewhere ( pfahl et al ., 1990 ). in general , 100 ng of reporter plasmid , 150 ng of β - galactosidase ( β - gal ) expression vector ( pch 110 , pharmacia ), and variable amounts of receptor expression vector were mixed with carrier dna ( bluescript ) to 1000 ng of total dna per plate . cat activity was normalized for transfection efficiency by the corresponding β - galactosidase activity ( pfahl et al ., 1990 ). cdnas for rxrα , rarα , rarβ , rarγ , trα , trβ , flag - rxrα , flag - trα , flag - rarγ and the deletion mutants cloned into pbluescript were transcribed by using t7 and t3 rna polymerases , and the transcripts were translated in the rabbit reticulocyte lysate system ( promega ) as described ( pfahl et al ., 1990 : zhang et al ., 1991b ). the relative amounts of the translated proteins was determined by separating the 35 s - methionine labelled proteins on sds - polyacrylamide gels , quantitating the amount of incorporated radioactivity and normalizing it relative to the content of methionine residues in each protein . in vitro synthesized flag - containing receptor proteins were checked for corrected sizes and antigenic specificity by immunoprecipitation with anit - flag antibody ( obtained from m . leahy , immunex , seattle , wash .) followed by sds - polyacrylamide gel electrophoresis . cdnas for rxrαm3 , rxrαm4 and rxrαm5 cloned into pbluescript were also translated in vitro . the translation start sites of these clones used the internal atg sequences at 28 , 61 and 198 amino acid position , respectively , as determined by the sds - page analysis of the 35 s - labelled translation products . to prepare trα and rarγ fusion proteins , flag - trα and 1 flag - rarγ cdnas were cloned in frame into the expression vector pgex - 2t ( pharmacia ). the proteins were expressed in bacteria using the procedure provided by the manufacturer . proteins were purified on a prepacked glutathione sepharose 4b column ( pharmacia ), and checked by gel retardation assays and western blot with anit - flag antibody . the tre used in the experiments was a 16 - bp perfect palindromic tre ( seq . id no : 4 ) ( tcaggtcatgacctga ) ( glass et al ., 1988 ). an oligonucleotide flanked by a bg1ii adaptor sequence was synthesized ( applied biosystems dna synthesizer ) and purified by polyacrylamide gel electrophoresis . oligonucleotides were annealed and were radioactively labeled using the klenow fragment of dna polymerase . tre / op is an oligonucleotide consisting of two tre half - sites with a 4 bp spacer ( seq id no : 5 ) ( gatcctgacctgagatctcaggtcag ). tre / half is an oligonucleotide consisting of one tre half - site ( seq id no : 6 ) ( gatctcaggtca ). βrare is the direct repeat of ra response element present in rarβ promoter ( seq id no : 7 ) ( agggttcaggcaaagttcac ). ere is the perfect palindromic er response element ( seq id no : 8 ) ( tcaggtcactgtgacctga ). these oligonucleotides are all synthesized with a bg1ii adaptor sequence . labeled dna probes were purified by gel electrophoresis and used for the gel retardation assay . cell extracts were prepared from cv - 1 cells in a buffer containing 20 mm hepes , ph 7 . 9 , 0 . 4 m kcl , 2 mm dtt and 20 % glycerol as described ( zhang et al ., 1991a ). in vitro translated receptor protein ( 1 to 5 μl depending on the translation efficiency ) was incubated with the 32 p - labeled oligonucleotides in a 20 - μl reaction mixture containing 10 mm hepes buffer , ph 7 . 9 , 50 mm kcl , 1 mm dtt , 2 . 5 mm mgcl , 10 % glycerol , and 1 μg of poly ( di - dc ) at 25 ° c . for 20 minutes . in general , relative low receptor concentration was used to obtain the clear effect of heterodimer formation . the reaction mixture was then loaded on a 5 % nondenaturing ployacrylamide gel containing 0 . 5 × tbe ( 1 × tbe = 0 . 089 m tris - borate , 0 . 089 m boric acid , and 0 . 002 m edta ). to analyze the effect of rxrα or the nuclear proteins on receptor dna binding activity , rxrα or the cell extracts were preincubated with receptor protein at room temperature for 10 minutes before performing the dna binding assay . when antibody was used , 1 μl of the antiserum was incubated with the specific translation products at room temperature for 45 minutes before performing the experiments described above . to analyze the interaction between rxrα and trα or and rarγ , purified flag - trα or flag - rarγ fusion proteins were loaded on the prepacked glutathione sepharose 4b columns . for control , the vector protein ( glutathione s - transferase ) prepared under the same conditions was also loaded on the separate columns . the columns were washed extensively with pbs with 1 % tritonx - 100 . 35 s - labelled in vitro synthesized rxrα , rxrαm4 and er proteins were applied to the columns . columns were then washed extensively with 3 times of 10 ml pbs . the bound protein was eluted with 50 μm tris ph 8 containing 50 μm tris ph 8 containing 5 μm glutathione . elutes were than concentrated by using a centricon 10 microconcentrator , and analyzed by denaturing polyacrylamide gels . twenty microliters of reticulocyte lysate containing in vitro translated 35 s - labelled rxrα were incubated with 5 μl ( approximately 0 . 2 μg ) of partially purified bacterially expressed flag - trα or flag - rarγ fusion proteins or similarly prepared glutathione transferase control protein in 100 μl buffer ( containing 50 mm kcl and 10 % glycerol ) for 15 min . at room temperature . when cross - linker was used , we added 2 μl of 100 mm dsp and continued the incubation at room temperature for 10 min . the reactions were then incubated with 1 μl of anti - flag antibody or preimmune serum for 2 hrs . on ice . immunocomplexes were precipitated by adding 60 μl of protein - a - sepharose slurry and mixing continuously in the cold room for 1 hr . protein - a - sepharose was saturated in tbs buffer ( tris - buffered saline ) or in ripa buffer when cross - linker was used . the immunocomplexes were washed four times with net - n buffer ( 20 mm tris , ph 8 . 0 , 100 mm nacl , 1 mm dtt , 0 . 5 % np - 40 ) or five times with ripa buffer when dsp was used , and resuspended in sds sample buffer containing 15 % β - mercaptoethanol , boiled and resolved by sds - polyacrylamide gel electrophoresis . the gels were fixed , dryed and visualized by autoradiography . previous data from us ( zhang et al ., 1991a ) and others ( lazar and berrodin , 1990 ; glass et al ., 1990 ; murray and towle , 1989 ; burnside et al ., 1990 ; rosen et al ., 1991 ) suggested that trs and rars bind more efficiently to their response elements by binding as heterodimers or heterooligomers . since proteins from nuclear extracts that enhance tr and / or rar dna binding have not been defined , we investigated the possibility that trs can bind with increased efficiency to the palindromic tre when complexed with other nuclear receptor proteins , in particular those that bind and activate the same or related response elements . using the gel retardation assay , we observed that trα bound to the tre as one major complex which migrates faster than the nonspecific band seen with unprogrammed reticulocyte lysate ( fig1 a ). this specific complex has been previously demonstrated to represent the binding of a trα monomer ( zhang et al ., 1991a ; forman and samuels , 1991 ). when trα was mixed with rxrα , a dramatic increase in dna binding was seen . a prominent complex which migrated slower than the nonspecific complex was observed while the faster migrating trα complex disappeared ( fig1 a ). the strong binding complex was observed at concentrations at which rxrα by itself did not form visible complexes with the tre ( fig1 d ). the effect of rxrα was specific since no significant increase in trα binding to the tre or change of trα binding pattern could be observed when it was mixed with rarα ( fig1 a ) or estrogen receptor ( er ) ( fig1 b ). in addition , when rxrα was mixed with er and labelled ere ( fig1 b ), no increased binding or slow electrophoretic mobility complex was seen . interestingly , when trα isoform trα - 2 was used , the formation of a low electrophoretic mobility complex was not observed either ( fig1 a ). these data suggest that trα and rxrα bind as heterodimers at least by an order of magnitude more effectively to the palindromic tre than by themselves . to investigate whether rxrα can also affect the binding of other nuclear receptors , rxrα was mixed with in vitro synthesized trβ , rarα , rarβand rarγ receptor proteins ( fig1 a ). similar to trα , the complex formed between trβ and tre was upshifted by rxrα . in the case of rarα , rarβ and rarγ , specific protein - dna complexes which migrated slower than the nonspecific complex were observed only in the presence of rxrα , while by themselves rars did not form detectable complexes with the tre under the conditions used . very similar results were also obtained with bacterially produced trα and rarγ ( data not shown ). the mobility of the slow migrating complexes formed between rxrα and trα was very similar to that formed between trα and nuclear protein ( s ) ( fig1 c ) from cv - 1 cells previously reported by us ( zhang et al ., 1991a ). this suggests that the protein ( s ) found in cv - 1 cells might be rxrα or related protein ( s ). next , we investigated the effect of t3 or ra on the interaction between rxrα and trs or rars ( fig1 d ). we observed no clear influence of these hormones on the formation of the slow migrating complexes when trα and rarα were studied although t 3 slightly increases the migration rate of the trα complex . similar results were also obtained when trβ , rarβ and rarγ were analyzed ( data not shown ). the observation that tr is upshifted by rxrα but not by rar and er and the fact that rar binds to the tre strongly only in the presence of rxrα but not of tr , strongly suggested that rxrα interacts with trs and rars to form heterodimers or larger complexes which interact very effectively with the palindromic tre . it is unlikely that rxrα catalyzed formation of tr and rar homodimers since at high trα concentrations , we have observed a trα dimer complex ( zhang et al ., 1991a ) which comigrates with the nonspecific band of the reticulocyte lysate , and which is at a different position from the complex we observed here in the presence of rxrα . the slow migrating complex observed here cannot represent the binding of rxrα homodimers either , since the migration of the complex is different depending on which receptor is mixed with rxrα ( fig1 a ). to examine more directly the components of the prominent upshifted complexes , we used rxrα , trα and rarγ derivatives that contained an eight - amino - acid epitope ( flag ) at the amino - terminal end of these receptors ( flag - rxrα , flag - trα and flag - rarγ , respectively ) which can be recognized by a specific monoclonal antibody ( hermann et al ., 1991 ; zhang et al ., 1991a ). the behavior of these receptor derivatives was indistinguishable from that of the wild - type receptor in both transcriptional activation ( zhang et al ., 1991c ; data not shown ) and dna binding activity ( fig2 ). when flag - rxrα was incubated with anti - flag antibody , we observed a specific complex ( fig2 a , lanes 14 and 23 ; fig2 b , lane 11 ; fig2 c , lane 13 ) which was not observed when preimmune serum was used ( fig2 b , lane 12 ). the complex may represent the binding of the antibody - catalyzed flag - rxrα homodimer or homooligomers . these data therefore suggest that rxrα by itself can not efficiently dimerize and bind to dna . similar antibody - induced dimerization has been observed for other receptors ( hermann et al ., 1991 ; zhang et al ., 1991a ). when flag - rxrα was incubated with trs and rars , it behaved essentially like rxrα , forming prominent slow migrating complexes ( fig2 a , lane 3 , 7 , 11 , 16 and 20 ). these complexes were strongly reduced when anti - flag antibody was added ( fig2 a , lanes 4 , 8 , 12 , 17 and 21 ). at the same time a higher molecular weight complex ( indicated by the solid triangle ) appeared . the reduction of the slow migrating complexes was observed when antibody was added either before or after both receptors were mixed ( data not shown ). the effect of antibody was specific in that the binding of these complexes was not changed when preimmune serum was used ( fig2 a , lanes 5 , 9 , 13 , 18 and 22 ), and the nonspecific binding of unprogrammed reticulocyte lysate ( indicated by open triangle ) was not affected by the antibody . in addition , the effect of anti - flag antibody was specific towards flag - rxrα since it did not influence the binding of trs and rars ( fig2 a , lanes 24 – 28 ) and rxrα ( fig2 b , lane 13 ). the migration of the faint higher molecular weight complex that appeared in the presence of antibody was dependent on the tr or rar isoform used . this complex migrated at the same position as the antibody - catalyzed rxrα homodimer ( fig2 a , lanes 14 and 23 ), suggesting that it may represent the binding of antibody - catalyzed flag - rxrα homooligomer . however , the intensity of this band was much weaker than the band observed in the absence of antibody . the inhibition of the slow migrating complexes in the presence of anti - flag antibody suggests that the antibody interacted with rxrα - trs or rxrα - rars complexes , resulting in the formation of larger complexes which have strongly reduced or altered affinity to dna . when trβ was assayed in the presence of flag - rxrα and anti - flag antibody ( fig2 a , lane 8 ), we clearly observed an additional complex ( indicated by arrow ). this complex migrated differently from the antibody - catalyzed flag - rxrα homodimer complex , and therefore may represent the binding of the antibody - upshifted flag - rxrα / trβ heterodimer . together , these data provide strong support for the assumption that the slow migrating complexes contain rxrα . more direct evidence comes from rxrα deletion mutant studies in which we show that the migration rate of the complexes depends on the size of the rxrα protein ( fig5 ). we show in fig1 that the slow migrating complexes migrate differently depending on which tr or rar isoform is mixed with rxrα , suggesting that the slow migrating complexes contain tr or rar . to directly test this , we used flag - trα and flag - rarγ ( fig2 b and 2 c ). for comparison , the effect of anti - flag antibody on flag - rxrα / trα and flag - rxrα / rarγ binding is shown on the same gel ( fig2 b , lanes 7 – 12 ; fig2 c , lane 8 – 13 ). the flag - trα behaved essentially as trα , forming one specific complex ( fig2 b , compare lane 1 and lane 7 ), which now can be upshifted by anti - flag antibody ( fig2 b , lane 5 ; indicated by arrow ) but not by preimmune serum ( lane 6 ). when flag - trα mixed with rxrα a slow migrating complex appeared ( lane 2 ) which is similar to the complex formed by trα and flag - rxrα ( lane 8 ). the appearance of this slow migrating complex was inhibited when anti - flag was added ( lane 3 ). the inhibition was specific since the binding was not affected when preimmune serum was used ( lane 4 ) and the nonspecific binding of unprogrammed reticulocyte lysate ( indicated by open triangle ) was not changed by the antibody . in addition , the antibody did not influence the binding of wild type rxrα or trα ( lane 13 and 14 ). similar to the effect of the antibody with flag - rxrα and trs or rars ( fig2 a ), we also observed the appearance of weak slow mobility complexes when incubating antibody together with flag - trα and rxrα . when flag - trα was replaced with flag - rarγ , similar inhibition effect of anti - flag antibody on flag - rarγ / rxrα binding was also seen ( fig2 c ). thus , taken together , these data strongly suggest that slow migrating prominent band observed in the presence of rxrα contain both rxrα and tr or rar . to investigate the dna sequence requirements for effective heterodimer binding , gel retardation assays were carried out using several tre related sequences : an inverted repeat of the tre ( tre / op ); a tre half - site ; the βrare , a retinoic acid response element ( hoffmann et al ., 1990 ; de thé et al ., 1990 ); and the estrogen response element ( ere , klein - hitpass et al , 1986 ) ( fig3 a ). rxrα alone did not bind to these dna sequences at the concentration we used . however , when it was mixed with trα , a specific slow migrating complex was observed on the tre , tre / op , and the βrare ( fig3 b ). similar to the palindromic tre , trα binds to the inverted repeat of the tre as one complex which was strongly enhanced and upshifted in the presence of rxrα . in case of the βrare , trα alone shows no visible binding but binds strongly when rxrα is present . binding of trα to the half - site is approximately as efficient as to the palindromic tre in the absence of rxrα . however , this binding was abolished in the presence of rxrα , suggesting that a trα / rxrα complex is formed but that a dimeric response element is required for heterodimer interaction . interestingly , trα also binds to the ere , a response element identical to the palindromic tre except for the 3 bp spacing in the center ( fig3 a ). however , when rxrα was added , the binding of trα to ere was abolished . the inhibition of trα binding to the ere by rxrα could also be due to the interaction between trα and rxrα in solution and formation of a heterodimer which has reduced affinity or does not bind to the ere . together these data demonstrate that a dimeric recognition sequence must be present for effective heterodimer binding and that heterodimer binding appears to be restricted to t 3 / ra responsive elements . our data suggest in addition , that rxrα can enhance receptor binding to quite different dimeric response elements , indicating a broad functional role for rxrα . the carboxyterminal end of tr and rar is necessary for interaction with rxrα to delineate regions of the trs and rars required for rxrα interaction , a number of trα and rarγ deletion mutants were investigated ( fig4 a , 4 c ). the deletion of 8 amino acids from the trα carboxyterminus did not affect trα - rxrα interaction , however , deletion of 197 amino acids ( trαm2 ) or 243 amino acids ( trαm3 ) abolished trα - rxrα complex formation . the trαm2 and m3 mutants bound effectively to the tre ( fig4 b ) as reported previously and are able to dimerize or oligomerize since several complexes can be observed ( zhang et al ., 1991a ). similar results were also observed with rarγ deletion mutants ( fig4 c , 4 d ). wild type rarγ and the mutants do not exhibit visible binding under the conditions used . as shown before , a strong dna binding complex was observed when rxrα protein was mixed with rarγ ( fig4 d ). however , deletion of 102 amino acid from the carboxyterminus ( rarγm1 ) completely abolished the binding of this complex . other carboxyterminal deletion mutants behaved similarly to the rarγm1 ( fig4 c , d ). our results on the tr mutants are consistent with our observation that trα - 2 which has an altered carboxyterminal region ( benbrook and pfahl , 1987 ) also does not form a low electrophoretic mobility complex with the tre in the presence of rxrα ( fig1 a ). mutational analysis of other receptors , including trβ , rarα and rarβ , revealed that the carboxyterminal region of these receptors is also important for their interaction with rxrα ( data not shown ). these results therefore indicate that the carboxyterminal region trα and rarγ is critical for interaction with rxrα . to delineate regions of rxrα required for nuclear receptor interaction , deletion mutants of rxr - α were investigated ( fig5 a ) for their ability to upshift trα and rarγ ( fig5 b , c ). deletion of 60 ( rxrαm1 ) or 75 ( rxrαm2 ) amino acids from the rxrα carboxyterminus abolished enhancement and upshift of the trα band while deletion of 28 ( rxrαm3 ) or 61 ( rxrαm4 ) amino acids from the amino terminus did not visibly affect interaction with trα , as analyzed by the gel retardation assay ( fig5 b ). however , a comparison of the complexes observed with rxrαm3 and rxrαm4 ( indicated by arrows ) clearly indicates that the size of the rxr protein determines migration of the complex . the smaller protein rxrαm4 forms a faster migrating complex than the larger protein ( rxrαm3 ). these data therefore provide direct evidence that rxrα participates in the complex . in addition , we observed that the carboxyterminal but not the aminoterminal end of rxrα is required for interaction with trα . interestingly , an internal deletion that spanned the hinge region and the aminoterminal half of the ligand binding domain ( rxrαm7 ) also abolished interaction with trα ( fig5 b ). thus both trs as well as rxrα require the carboxyterminal domain for heterodimer formation . in addition , however , a truncated rxrα form ( rxrαm5 ) in which the aminoterminal 198 amino acids were deleted ( including the dna binding domain ) also failed to form a complex with trα . a second mutant lacking the dna binding domain , rxrαm6 , was also unable to upshift trα . the absence of a complex and the fact that trαdna binding was not inhibited , suggest that portions of the dna binding region of rxrα are required for interaction with trα . when we replaced trα with rarγ identical results were obtained with the rxrα mutants ( fig5 c ). in this experiment , rxrαm4 also forms a complex with rarγ which migrates faster than the complex formed by rxrαm3 and rarγ , as indicated by arrows . similar results were also obtained when trβ , rarα and rarβ were used ( data not shown ). these data thus support the hypothesis that interaction of rxrα with trs and rars is mediated by the same structural determinants . the ability of rxrα to enhance rar and tr dna binding could also allow enhancement of transcriptional activation of these receptors on the tre , a known ra response element ( graupner et al ., 1989 ; umesono et al ., 1988 ). when low concentrations of rxrα expression vector were cotransfected with rarα and the tre 2 - tk - cat reporter construct , a strong enhancement of the rarα activity was observed ( fig6 a ). most interestingly , this strong enhancing activity by rxrα was seen at ra concentrations ( 10 - 7 m ) at which rxrα by itself was only slightly activated ( fig6 b ). the increased activation of the reporter gene in the presence of both retinoid receptors is clearly more than additive at certain receptor concentrations as shown . for instance , when 25 ng of rarα and 25 ng of rxrα expression vectors were used , a strong synergistic effect was observed . a very similar enhancing effect was also observed with rarγ ( fig6 b ). in this study , we analyzed the effect of rxrα on rarγ activity under several ra concentrations using the tre - tk - cat construct as reporter . at the concentrations used , neither rxrα by itself nor rarγ by itself could elicit any significant transcriptional response at ra concentrations between 10 - 9 m and 10 - 7 m ( fig6 b ). however , when they were transfected together , a synergistic effect ( 2 to 4 fold induction ) was observed over this ra concentration range . thus , the ability of rxrα to enhance rar dna binding in vitro correlates with an enhanced transcriptional activation capacity of rxrα - rar complexes in vivo . the above experiment did not allow to determine whether rxrα itself requires ligand binding to boost transcriptional activation with rarα or rarγ . when we cotransfected rxrα with trα , we also observed synergism between both receptors on \ tre - tk - cat reporter constructs ( fig7 ). some synergism was observed when only thyroid hormone ( t 3 ) was added , while optimal synergism required the presence of both ligands , t 3 and ra . remarkably , only low amounts of rxrα as well as trα were required to observe a strong activation of the reporter genes . to examine in detail the ligand requirements for the putative rxrα / trα complex , we compared the ra concentrations required to activate rxrα alone or in combination with trα . rxrα expression vector alone or together with trα expression vector were cotransfected into cv - 1 cells with reporter constructs that contain either a single ( tre - tk - cat ) or double ( tre 2 - tk - cat ) response element . cells were grown in the presence of a constant amount of t 3 ( 10 - 7 m ) and various amounts of ra ( 10 - 10 to 10 - 5 m ). we observed a dramatic shift of the ra responsiveness of rxrα in the presence of trα . in cases of both the single tre and the double tre reporter , the rxrα sensitivity to ra appeared to be increased by at least 2 orders of magnitude ( fig7 a , b ). this enhanced ligand sensitivity is not due to the activation of endogenous rars by trα since no effect of cat activity was observed when trα was transfected alone ( fig7 a , b ) consistent with previous observations ( graupner et al ., 1989 ). trα alone at his low concentration did not induce the reporter gene to a high degree in the presence of t 3 , although an approximately 10 fold induction was observed ( this is difficult to see on the scale used in fig7 ). thus , while rxrα boosts dna binding and transcriptional activation of other receptors , by forming a complex with trα , its own ligand affinity is also dramatically increased in the heterodimer complex . our observation that rxrα exerts its effect on rarα and rarγ transcriptional activity in the presence of less than 10 - 7 m ra , suggests that complex formation between rxrα and rarα or rarγ also boosts the ligand sensitivity of rxrα and that ra may be a natural ligand for rxrα . an important question is whether rxrα can form heterodimers with the other receptors in solution or whether the heterodimeric complexes are only formed in the presence of specific dna sequences . the ability of rxrα to interact with other receptors in the absence of dna could be expected to largely enhance the efficacy of rxrα as a regulator of heterologous receptor activity . to investigate interaction between rxrα and tr or rar in the absence of dna , we took advantage of a unique affinity column containing glutathione coupled to sepharose to which bacterially produced receptor - glutathione transferase fusion protein binds specifically and can be eluted with free glutathione ( smith and johnson , 1988 ). trα or rarγcdna were cloned into the prokaryotic expression vector pgex - 2t , and expressed as trα - or rarγ - glutathione transferase fusion proteins in bacteria . the fusion proteins were able to interact with in vitro synthesized rxrα as determined by gel retardation ( data not shown ). we used bacterially produced trα - or rarγ - glutathione transferase bound to the affinity resin and mixed this with in vitro synthesized 35 s labelled receptors . after extensive washing , labelled rxrα could be specifically eluted with glutathione from a column that contained bound trα or rarγ fusion protein , but rxrα was not retained on a column that contained only bound glutathione transferase ( fig8 ). labelled er was not retained on by the trα or rarγ fusion proteins , while the mutant rxrαm4 that lacked 61 amino acids at the amino terminus and was able to upshift trα and rarγ , was retained . to further document the physical interaction between rxrα and tr or rar , we incubated labelled rxrα protein , produced by cell - free translation , with or without bacterially produced flag - trα or flag - rarγ proteins . anti - flag antibody was used to examine whether rxrα could be precipitated together with flag - trα or flag - rarγ . as shown in fig8 b , precipitation of flag - trα or flag - rarγ resulted in a significant coprecipitation of labelled rxrα protein . the coprecipitation occurred even in the absence of cross - linker while it was largely enhanced when cross - linker ( dsp ) was used . the coprecipitation is specific since no significant amount of labelled rxrα was precipitated when preimmune serum was used or when rxrα was incubated with nonspecific control protein together with the anit - flag antibody . the observation that rxrα could be coprecipitated in the absence of cross - linker and the results obtained with the affinity column strongly suggest that rxrα forms a stable complex with either tr or rar in solution , and supports our interpretation of the gel retardation results shown in fig2 . several lines of evidence are provided here for the direct interaction between rxrα and trs or rars , which result in the formation of heterodimers which exhibit strong dna binding to a number of t3 / ra dimeric response elements . first , when rxrα was mixed with trα , trβ , rarα , rarβ or rarγ , a prominent slow migrating complex was formed which migrated at different positions depending on which tr or rar was used ( fig1 a ). second , using antibodies against rxrα , tr and rar , we demonstrate that the binding of these slow migrating complexes can be dramatically altered by these antibodies ( fig2 ). third , rxrα mutational analysis shows that the migration of these complexes depended on the size of the rxrα protein ( fig5 ). finally , a study using affinity column chromatography and immuno - coprecipitation results demonstrate that rxrα can interact with tr and rar in the absence of dna ( fig8 ). the enhancement of dna binding and the characteristic upshift observed for all receptors in the presence of rxrα are very similar to the enhanced dna binding and upshifts of trα observed in the presence of nuclear extract from several cell lines ( fig1 c ; zhang et al ., 1991a ). in addition , all trα mutants investigated behave virtually identical with rxrα and nuclear extract protein ( fig4 ; zhang et al ., 1991a ). it is therefore quite possible that rxrα is identical or closely related to the cellular protein previously described ( lazar and berrodin , 1990 ; murray and towle , 1989 ; burnside et al ., 1990 ). according to its enhancing effects on tr dna binding , the nuclear protein or proteins that enhance tr dna binding have been termed tr auxiliary proteins — trap ( reviewed by rosen et al ., 1991 ). however , if these proteins are identical with rxrα or a related isoform , this nomenclature is insufficient to describe their function . rxrα , as an example of this new receptor subclass , can not dimerize by itself efficiently but can interact with trs or rars to form heterodimers with strong dna binding activity . tr and rar as well as rxrα require regions near the carboxyterminal end for interaction ( fig4 and fig5 ). interestingly , these regions are also required for tr interaction with cjun , a component of the transcription factor ap - 1 that has recently been shown by us to regulate tr and rar activities ( zhang et al ., 1991c ; yang - yen et al ., 1991 ). thus , this carboxyterminal region may be viewed as a domain that can interfere with other active protein regions in possibly both cis and trans locations . the ligand binding domain of trs and rars was shown to possess 9 heptad repeats of hydrophobic amino acids which are structurally similar to the leucine - zipper dimerization domain ( forman et al ., 1990 ). these leucine - zipper like motifs are thought to mediate the receptor dimerization activity by a coiled - coil α helix in which a hydrophobic surface along one side of the helix could act as a dimerization interface . similar heptad repeats are also present in the ligand binding domain of rxrα . the requirement of the ligand binding domain of both rxrα and tr or rar for heterodimer formation implicates these leucine - zipper like motifs in the direct interaction between both receptors . however , rxrα may possess some other special structural features which are not present in tr or rar since we could not observe clear interaction between tr and rar when they were mixed together and were assayed under the same conditions ( fig1 a ). these special structural features may not allow rxrα homodimer formation , but allow rxrα to efficiently interact with tr and rar . a detailed mutational analysis of rxrα receptor protein is therefore important in order to understand the mechanism of interaction between rxrα and tr or rar . while the heptad repeats in the ligand binding domain of rar , tr and rxrα may effectively interact with each other , and thereby allow receptor contact , at the same time the interaction may be extended through the dimerization domain embedded in the dna binding region of nuclear receptors ( zhang et al ., 1991a ; härd et al ., 1990 ; luisi et al ., 1991 ; schwabe et al ., 1990 ). this is supported by our observation that the dna binding domain of rxrα is also important for efficient interaction with tr or rar ( fig5 ). trs and rars are important mediators of cellular development and differentiation processes . the observation that rxrα can interact with trs and rars in the absence of dna ( fig8 ) and the fact that the heterodimer can bind to a number of t3 / ra specific response elements ( fig3 ) point to the profound role of rxrα in regulating these cellular processes . although interaction between rxrα and tr or rar occurs in solution , the outcome of this interaction may depend on the sequence of the response element in particular genes . in other words , the specificity and the extent of transcriptional regulation , either positively or negatively , by receptor interactions maybe largely determined by the nature of the response elements and the receptors ( and their concentrations ) with which rxrα interacts . synergistic transcriptional activity of rar and tr on the palindromic tre was observed when they were cotransfected with rxrα ( fig6 and fig7 ). these in vivo observations correlate very well with the strong dna binding of heterodimers formed between rxrα and tr or rxrα and rar . interestingly , considerable enhancing activity of rxrα is observed in the absence of ra while optimal enhancement occurs already at low ra concentrations ( less than 10 - 7 m ), whereas higher ra concentrations are required to activate rxrα alone ( more than 10 - 6 m ; fig6 , fig7 ; mangelsdorf et al ., 1990 ). thus , while rxrα boosts very efficiently the activity of trs and rars in terms of dna binding and transcriptional activation , its own ligand responsiveness is also boosted by the heterodimerization , i . e . mutual enhancement is occurring . this is most likely one of the major roles of rxr . we like to call this novel activity a “ booster receptor ” ( b - receptor ), in contrast to activator receptors ( a - receptors ). in addition to its enhancer activity , rxr forms a complex with trα ( and trβ , data not shown ) that appears to require two distinct hormones for full activation . this novel type of receptor complex allows direct cross - talk between two different hormonal signals at the receptor level . the palindromic tre analyzed here is derived from the growth hormone ( gh ) tre . two years ago , bedo et al . ( 1989 ) reported that the gh gene can be induced by ra and that the presence of t 3 increases the effectivity of ra by close to 3 orders of magnitude ( from 10 - 6 m to 10 - 9 m for optimal induction ). this type of in vivo observation is very similar to ours , where 10 - 5 m ra are required for rxr activation while 10 - 8 m is sufficient for activation of rxrα in the presence of trα and t 3 . a comparable synergistic effect has recently also been reported for the induction of granulocyte differentiation in leukemic cells including hl60 ( ballerini et al ., 1991 ). because low concentrations of rxrα are sufficient for boosting rarα and trα activity , an extremely sensitive regulatory mechanism is created that can respond very efficiently to small changes in the concentrations of individual components . our data suggest that , contrary to earlier suggestions ( mangelsdorf et al ., 1990 ), ra is an important natural ligand for rxrα ; whether other natural retinoids exist that effectively activate rxrα homodimers at physiological concentrations remains to be determined . at present it appears that more than one rxr subtype exists ( rxrα and rxrβ ) that may have distinct booster specificities . even the same rxr subtype may show considerable selectivity depending on the response element ( fig3 ), interacting receptor and receptor concentration ( fig6 a ). we have provided evidence here that the booster capacity for rxrα towards trα is much higher than towards rarγ ( fig6 and fig7 ), and effective over a wider receptor concentration range as well ( data not shown ). the subfamily of b - receptors may also include a substantial number of orphan receptors for which no specific ligands could be detected so far or other receptors that require very high ligand concentrations for efficient activation . since rxrs appears to be encoded by more than one gene ( mangelsdorf et al ., 1990 ; hamada et al ., 1989 (, rxrβ whose dna and ligand binding domains are almost identical to those of rxrα is an equally good candidate . in general , the mechanisms of heterodimer formation is widely used by transcription factors , the most well known examples being ap - 1 ( reviewed by karin , 1990 ) and the more recently described myc - max heterodimeric ( blackwood and eisenman , 1991 ). because of the obvious advantage of heterodimeric and booster receptors in many systems , our studies presented here may be the tip of the iceberg of a large field of receptor action not yet explored . ballerini p ., lenoble , m ., balitrand , n ., schaison , g ., najean , y ., and chomienne , c . 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