The Unique C-Termini of the Thyroid Hormone Receptor Variant, c-erbAa2, and Thyroid Hormone Receptor 4 Mediate Different DNA-Binding and Heterodimerization Properties
Deborah
Katz, Thomas
J. Berrodin,
Departments of Medicine University of Pennsylvania Philadelphia, Pennsylvania
and Mitchell
A. Lazar
and Human Genetics School of Medicine 19104
INTRODUCTION
Thyroid hormone receptors (TRs) mediate the regulation of gene transcription by thyroid hormone (T3) by binding to TS-responsive elements (TREs) in target genes. c-erbAa2 is a C-terminal TR variant which does not bind T3 and is a dominant inhibitor of T3 action. When synthesized in Escherichia Co/i, (r2 formed two TRE-binding complexes similar to the monomeric and homodimeric forms of TRal. However, a2 did not bind nearly as well as TRal. Furthermore, ~y2 failed to bind DNA with proteins that heterodimerized with TRal. TRal and (~2 also did not bind DNA as heterodimers with one another. The differences between TRal and a2 were further analyzed by studying a variety of C-terminal mutants synthesized in reticulocyte lysates. Deletion of the last 20 of the 122 unique amino acids (aa) of (r2 increased its DNA binding to approximately the level of TRal, indicating that the C-terminus of (~2 is an inhibitory domain. This (~2 mutant ((u2AC) was still unable to heterodimerize with nuclear proteins, as were C-terminal deletion mutants of TRal. We hypothesized that fusion of TRcul-specific sequences to the C-terminus of cy2AC would transfer the property of heterodimerization. Indeed, although (u2/(~1 chimeras containing the last 40 and 70 aa of TRal failed to heterodimerize with nuclear proteins, addition of the last 100 or 150 aa of TRal did render a2AC heterodimerization competent. Thus, TRal contains a C-terminal structure which is necessary for heterodimerization and can confer this property on (r2, which lacks this domain. The effects of the unique C-termini of TRal and (r2 on their in vitro DNA binding have important implications for their mechanisms of action in viva. (Molecular Endocrinology 6: 605-814, 1992) 088&8809/92/0805-0814$03 00/o Molecular Endocrinology Copyright 0 1992 by The Endcmne
Thyroid hormone (T3) regulates gene transcription by interacting with nuclear T3 receptors (TRs) (l-4). The specificity of this process is determined in part by interactions between TRs and TS-responsive elements (TREs) in target genes. The complexity of T3 action has been underscored by the discovery that there are one cy and two p TR forms (5-l 3) which are expressed in a tissue-specific manner (5, 10, 14, 15) and differentially regulated during development (16, 17) and by a variety of hormonal (5,16-l 9) and pharmacological (20, 21) manipulations. Furthermore, the TRs can bind to TREs as monomers and dimers (22, 23) as heterodimers with other nuclear receptors such as the retinoic acid receptor (RAR) (24, 25) and as heteromeric complexes with one or more as yet unidentified nuclear proteins (23, 25-31). The TR-nuclear protein heterodimer is the preferred TRE-binding form of the TR in vitro, due to the increased stability of this complex (23). c-erbAa2 (referred to hereafter as ~y2)is an alternative splicing product of the TRa gene (9-12, 32, 33). In the rat, cu2 and TRal are identical for 370 amino acids (aa) but have completely different C-termini; TRcul contains an additional 40 aa which are identical to those in the TR/J forms, whereas (~2 has a unique l22-aa extension which is incapable of binding T3. ~y2 mRNA and protein are normally expressed at high levels in a variety of tissues (10, 14, 34). Functionally, ot2 can inhibit the T3dependent transcriptional effects of the bona fide TRs (35, 36) and is similar in this respect to the oncoprotein v-erbA (37-40) as well as to C-terminal mutants of TRP which cause inherited syndromes of generalized T3 resistance (41-43). The mechanism of action of c~2 is poorly understood. cu2 could compete with the TR for binding to TREs or form inactive heterodimers with TRs at the exclusion of homodimeric or other heterodimeric TR forms which are transcriptionally active. Formation of inactive heterodimers has also been de-
Somty
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MOL
ENDO.
1992
Vol6
No. 5
806
scribed for the few other transcription factors with inhibitory proteins known to be encoded on the same gene, such as bovine papillomavirusproteins E2/E2TRand E8/E2 (44,45) and LAP/LIP (liver transcriptional activator and inhibitor, respectively) (46). Since (~2 is structurally similarto the TRs, it could also compete for bindingto the proteins which specifically enhance TR bindingby heterodimerization,analogousto the inhibition of AP-1 transcription factor Jun/Fos by the related inhibitor L?rFosB (47). Finally, a2 could act by sequestering other transcriptionfactors which contribute to T3 action. To determinehow the structural differencesbetween TRal and ot2 affect their function, we have compared their DNA-bindingpropertiesin vitro. We show that the uniqueC-terminusof ~y2reducesits DNA binding,which can be restored to the level of TRal by smalldeletions in this region. Furthermore, a2 does not bind DNA as a heterodimerwith nuclear proteins which enhance TR binding because it lacks the C-terminus of the TR. However, fusion of the TR C-terminusto a2 creates a chimeric protein which is able to heterodimerize with nuclearproteins. Thus, C-terminaldifferencesbetween TRal and a2 have functional significancein vitro which have important implicationsfor our conception of how the TR and a2 regulateT3 action.
RESULTS
Wild type TRal and a2 were expressed in fscherichia co/i fused at their N-termini to 17 aa containing an epitope recognized by a commerciallyavailable monoclonal antibody (FLAG; see Materials and Methods). Figure 1A shows the structure of the recombinant proteins, highlightingthe identity of TRal and 012until their C-termini. TRal contains 40 C-terminalaa which areidenticalto those of other TRs, whereas012contains a unique 122-aa C-terminus. Shown in Fig. 1B is a Western blot analysisof soluble preparationsof these proteins, using the FLAG antibody for detection. Both TRal and 012were of the predicted size [49 and 57 kilodaltons(kDa), respectively, including the 2-kDa Nterminal addition]. Coomassieblue staining revealed the recombinantprotein to compriseabout 0.5% of the total soluble protein in each case, consistent with an estimatebasedupon the T3 bindingof the TRal preparation(data not shown).A closely spaceddoublet was observed in some preparations of a2, with the faster migrating species always less abundant. Figure 1C shows that similar results were obtained by Western analysis with a monoclonal antibody raised against TRoll, which recognizes an epitope between amino acids 123-370 of TRoll , since it detected TRcul and a2, as well as a glutathione-S-transferase-TRotlfusion proteincontainingaminoacids 123-410 of TRal . Thus, no C-terminalproteolytic products, which could potentially inhibit DNA bindingand dimerizationof the protein (39) were detected in the TRal preparation, and the major (~2specieswas the full-length protein as well.
A TRal 370
492
c-erbAcr2
B
FLAG
Ab
C
TRal
Ab
-200
-200
-97 -69
-97
/
-46
X‘PM **
’ -69 -46
-30 -21
-30 -21
Fig. 1. TRL~I and c-erbAcu2 Proteins A, Schematic structure of the proteins. The 17-aa N-terminal addition contains the FLAG epitope and is detailed in Materials and Methods. The positions of the unique regions are numbered as in the wild type proteins. B, Western analysis of E. co/i-synthesized proteins with FLAG antibody. Twenty micrograms of soluble protein preparations were used in each case. C, Western analysis of E. co/i synthesized proteins with antiTRa monoclonal antibody. Twenty micrograms of soluble protein preparations were used in each case. Also shown for comparison is a glutathione-S-transferase-TRal ligand-binding domain (123-410) fusion protein [GST-TR(LBD)] whose predicted mol wt is 59K. Migration of mol wt standards (in thousands) is indicated.
The DNA binding properties of the TRal and c~2 proteinswere studied in the gel electrophoreticmobility shift assay, usingthe palindromicTRE, TREp (23) and the proteinsshown in the Western analysisabove. Fig. 2A shows that TRclll formed two majorcomplexes(lane l), which we have previously identified as monomeric and homodimericon the basisof their relative mobilities as well as methylation interference and mixing experiments (23). ~y2also formed two major TREp-binding complexes (Fig. 2A, lanes 2 and 3) each retarded relative to the correspondingTRal complex consistent with the larger size of a2. However, despite the fact that approximately equal amounts of the TRotl and 012 proteins were used in this experiment (Fig. 2A, lanes1 and 2) densitometric analysis of the complexes revealed that the binding of 012was approximately 5% that of TRal. A 4-fold increase in 012concentration resulted in increasedbinding (Fig. 2A, lane 3).
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DNA Binding by Thyroid Hormone Receptor and Variant
Ab Liver TRal
+++
+
NE
+++
5
2
2
+
222
11
5 20
C-WbAOZ
123
807
1 2
3
4 5
6
5
5 5
7
8
51020
9 101112
Fig. 2. DNA Binding of TRorl and c-erbAa2 A, Direct comparison of TFM and c~2. Equal amounts (5 Kg) of the same preparations as shown in Fig. 1, B and C, were used in the binding reactions for lanes 1 and 2, with a 4fold excess of 012in lane 3. B, Heterodimerization potential of TRal and 012.The E. co/i-synthesized proteins were incubated with TREp, alone or in combination with one another, liver extract, or FLAG antibody as indicated. Lanes 1 and 4, No TR or c-erbAa2; lanes 2, 3, 5, 6, and 7, 2 pg TRcz~ ; lanes 11 and 12,l pg TRal ; lanes 7,8,9, and 10,5 pg c-erbAol2; lane 11, 10 rg c-erbAa2; lane 12, 20 rg c-erbAa2; lanes 3, 4, 5, and 9,5 pg liver nuclear extract (NE); lanes 2,3, and 10,l /II FLAG antibody (Ab). The autoradiograph in A was exposed to x-ray film for 16 h, that in B for 24 h. Rapidly migrating complexes which supershift with FLAG antibody may represent minor DNA-binding fragments of TRcJ and a2.
The abilities of TRoll and ot2 to interact with a TRbinding nuclear protein and with each other were also studied, as shown in Fig. 2B. TREp binding of TRal
was again greater than (~2,despite the fact that more 012was used in this experiment (i.e. 2.5-fold more cu2 than TRcx~in lanes 8 and 6, respectively). Addition of liver nuclearextract enhancedthe bindingof TRal due to formation of a novel complex which migratedfaster than the TR homodimer complex (Fig. 2B, compare lanes5 and 6). We have previously shown that this new complex is due to heterodimerizationbetween the TR and a 42-kDa protein in the liver nuclear extract (23). In contrast, (~2did not heterodimerizewith this TR-binding protein, and addition of liver extract had no effect on (~2binding (Fig. 28, compare lanes 8 and 9). Addition of FLAG antibody supershiftedall TRal and (~2complexes, includingthe TRoll heterodimer(Fig 28; TRotl , lanes2 and 3; (~2,lane lo), confirmingthe involvement of the recombinantproteins.However, mixture of TRLu~ and ot2 at varying molar ratios resulted in no additional complexes(Fig. 2B, lanes7,11, and 12); since a TRcul/ (~2heterodimershouldhave been detectable as a complex migratingbetween TRal and cu2,these data indicate that the two proteins did not form DNA-binding
heterodimers. It is possible that the proteins formed non-DNA-binding heterodimers, but this must have been a relatively weak interaction because even at a 20-fold molar excess of 012the binding of TRal was only modestly decreased(Fig. 2B, lane 12). Thus while both TRal and ot2 bound TREp, a2 did not bind as well as TRal and did not bind as a heterodimerwith the TR-binding protein in liver nor with TRal. In order to examine the differences between TRcvl and a2 in more detail, the wild type proteins and a variety of C-terminaldeletionmutantswere synthesized in reticulocyte lysates (without N-terminal FLAG sequences).The translationswere performedin the presence of [35S]methionineand, as shown in Fig. 3A, the full-lengthproteins were predominantin each case. We have previously noted that TRal forms primarily a single,monomericTREp-bindingcomplexwhen synthesized in this manner, due in part to the lesseramounts of protein made (23). This was confirmed in the experiment shown in Fig. 3B, which shows that deletion of 64, 85, 160, and 210 aa (labeledotAC-1, aAC-2, LuAC, and o(AC-3, respectively) from the C-terminusof TRal did not interfere with TREp binding,although the migration of the complexes increased as the proteins decreased in size. These C-terminal deletion mutants of TRoll contain none of the TRal-specific C-terminal portion and are thus identical to corresponding deletions of cu2.In contrast, and in generalagreementwith the results using bacterially synthesized proteins, wild type 012bound DNA much less well than any of the truncation products or wild type TRal However, deletion of 84 ((Y~AC-2), 38 (a2ACl), or as few as 20 (o12AC)aa of the unique ot2 C-terminusrestored TREp binding to approximately the level of TRal. Thus the C-terminusof cu2inhibited DNA binding by the rest of the molecule,but this inhibitory domainwas interrupted by eliminationof the last 20 aa. We next assessedthe ability of the reticulocyte lysate synthesized proteins to form heterodimerswith nuclear proteins. Figure 4A shows that, as expected, formation of a heterodimericcomplex with the TR-bindingprotein in liver enhanced DNA binding by wild type TRotl. Truncation of the C-terminus of TRotl abolishedthe effect of liver protein, as was previously noted with the proteins present in GH3 and JEG3 cells(27), indicating that the C-terminusof TRal is necessaryfor heterodimerization. Again, DNA binding by wild type 012was difficult to detect and was not stimulated by liver extract, in agreement with earlier results (Fig. 28). Similarly, although the 20-aa deletionmutant (a2AC) bound TREp well, its binding was not enhanced by liver extract, and there was no evidencefor heterodimerization. The same was true for the TRcwl C-terminal deletion mutant lacking 64 aa (data not shown). Note that in Fig. 4A and in later experiments(Fig. 5B), TRLu~formed a slowly migratingcomplex in addition to the prominent monomericcomplex. The migrationof this complex was indistinguishablefrom the heterodimer formed by the TR and a protein present in JEG3 cells (27, 30). We hypothesized that this additional complex, which was
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MOL 808
ENDO.
1992
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No. 5
al aAC-1 aAC-2 aAC
E 370
492
a2 472
a2AC a2AC-3 a2AC-2
Fig. 3. DNA Binding of TRal , c-erbAa2, and C-Terminal Deletion Mutants A, Expression and structure of reticulocyte lysate-synthesized proteins. SDS-PAGE analysis of %-labeled protein. Schematized structures of the proteins are shown at right for comparison. Mol wt (in thousands) of standard proteins electrophoresed in the leftmost lane is indicated. Equal amounts of TCA-precipitable counts, adjusted for methionine content, were applied to each lane. B, DNA binding. Equal amounts of the reticulocyte lysate-synthesized proteins were incubated with TREp. Arrows indicate approximate positions of specific complexes. aAC-3 is a 211 -aa C-terminal truncation product of Tkl A rapidly migrating nonspecific complex was observed in binding reactions in this experiment, including that with unprogrammed lysate (I).
present in some but not all experiments, was due to the presenceof nucleated red blood cell precursors in somebatchesof reticulocyte lysate. Indeed, when bacterially synthesized TRoll was mixed with unprogrammedreticulocyte lysate, we observed a heterodimerit complex which migrated slower than the TR homodimer,as shown in Fig. 48. The above data suggestedthat the C-terminusof ot2 either lacked a heterodimerization domain present in TRal or actively prevented heterodimerization.In order
to distinguishbetween these two possibilities,we synthesized a seriesof chimeric proteinsin which increasing lengthsof the TRcul C-terminuswere fused to (~211C (Fig. 5A). Note that since only 40 aa are unique to TRcul, fusion proteins of increasing length contained repeatsof sequencescommonto TRal and a2. Nevertheless, Fig. 5B (rap panel) shows that a chimeric cu2AC/crl protein containingthe 40 aa uniqueto T&l (labeled0l2AC/oll-40 in the figure) bound TREp but did not heterodimerize (Fig. 58, lanes 7 and 8) and a
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DNA Binding by Thyroid Hormone Receptor and Variant
809
B
A al Nuclear Extract
aAC
a2
TR++RL - + +
a2AC
-+-+-+-+ \
Fig. 4. Heteroclimerization Properties of TR& , c-erbAa2, and C-Terminal Deletion Mutants A, The proteins shown in Fig. 3A were incubated in the presence or absence of liver nuclear extract (2 pg) in the electrophoretic mobility shift assay. An additional band due to heterodimerization in the presence of nuclear extract is seen only with TRai The positions of the monomer (M) and heterodimer (HD) complexes are indicated by arrows.The asterisk indicates the position of a complex in the TRcx~ lane which is due to the presence of a heterodimerizing species in unprogrammed reticulocyte lysate, as shown in B. B, Bacterially synthesized TRal (5 pg) was incubated with and without 5 ~1 unprogrammed reticulcyte lysate. A novel TR heterodimer (HD) complex which migrated slower than the TR homodimer (D) was only observed when the TR and lysate were mixed.
chimeric protein containing the 70 C-terminal aa of T&l (a2AC/al-70) was similarly unable to interact with nuclear proteins (Fig. 5B, lanes 9 and 10). However, chimeric proteins with the last 100 (cu2AC/c~l100) or 150 (cx~AC/(Y~ -150) aa of TRotl fused to cu2AC not only bound TREp but did heterodimerize with the liver TR-binding protein (Fig. 58, lanes 11-l 4). Interestingly, a more greatly retarded TREp-binding complex was detected in the absence of nuclear extract only with the proteins which interacted with the liver TRbinding protein, suggesting that the heterodimerizing protein present in this batch of reticulocyte lysate interacted with a similar domain. The experiment shown in the top panel of Fig. 58 used 32P-labeled TREp to detect DNA-binding complexes. The lower panel of Fig. 5B shows the results of separate experiments in which unlabeled TREp was used along with 35S-labeled proteins in order to track the migration of in vitro synthesized proteins themselves. Again, TRal and the a2AC/al chimeras containing the last 100 or 150 aa of TRoll were the only proteins to form heterodimeric complexes in the presence of nuclear extract (Fig. 5B, lanes 11-l 4). These data indicate that (~2 is lacking a heterodimerization domain contained in TRal , but that the C-terminal 40 aa of TRal, while necessary, are part of a longer domain which must remain continuous in order for the protein-protein interaction to occur.
DISCUSSION One of the most unique aspects of T3 action is the existence of (~2, an endogenous dominant inhibitor of TR-mediated effects. The present work confirms other studies demonstrating that ot2 does not bind DNA as well as TRal , even though it contains an identical DNAbinding domain (10, 48). The reduced DNA binding of a2 is apparent with preparations of the protein synthesized in bacteria as well as in reticulocyte lysates. Our inability to reproducibly detect binding of reticulocyte lysate-synthesized (~2 may be due to the low levels of a2 protein relative to that synthesized in E. co/i, just as the DNA binding and homodimerization of TRcwl were more easily demonstrable in this system (23). However, truncation of 20 aa from the C-terminus of a2 increased the DNA-binding of this reticulocyte lysate-derived protein to the level of wild type T&l. It is therefore likely that the C-terminal of 012 adversely effects its DNA binding. This hypothesis is supported by the observation that a variety of C-terminal deletion mutants of TRal or ot2 bind DNA about as well as does wild type TRcul. This suggests that the intact C-terminus of c~2 alters the shape of the protein in such a way that the DNA-binding domain either has reduced access to DNA or is itself in a conformation less favorably disposed for DNA binding. The unique region of cu2 has the same pl as that of TRcul , but is much more hydrophilic (by KyteDoolittle analysis). It also is extremely serine-rich (15% in the rat, 12% in the human), and the possibility exists
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MOL ENDO. 1992 610
Vol6 No. 5
l *+
cHD
HD+ M-e
I
2
3
I
5
6
7
*
9
III
11
12
13
1.
Fig. 5. DNA Binding and Heterodimerization Properties of T&l, c-erbAa2, and Chimeric Proteins A, Expression and structure of reticulocyte lysate-synthesized proteins. SDS-PAGE analysis of 35S-labeled protein. Mol wt (in thousands) of standard proteins electrophoresed in the leftmost lane is indicated. Schematized structures of the proteins are shown for comparison at right. B, DNA binding and heterodimerization. Arrows indicate the position of heterodimeric (HD) and monomeric (M) complexes. The asterisk indicates the position of the heterodimeric complex containing the TR and the reticulocyte lysate protein, only observed with those proteins (al, &AC/al -100, and c~2AC/~~l-l50) which also heterodimerized with the liver protein. Top panel, 32P-labeled TREp; bottom pane/, unlabeled TREp, 35S-labeled proteins. Double asterisks indicate the position of a 35Slabeled protein present in the unprogrammed reticuloctye lysate which migrated to this position even in the absence of DNA.
that phosphorylation or some other posttranslational modification of the C-terminus of ot2 regulates its ability to inhibit DNA binding in viva. Although the inhibitory domain can be disrupted by deleting just 20 aa, the unique 122 aa of cu2 is not sufficient because a chimeric protein in which this polypeptide has been fused to the C-terminus of TRal binds DNA as well as TRotl does (D. Katz and M. Lazar, preliminary results). Thus, the
domains responsible for the inhibitory function of the (~2 C-terminus as well as the heterodimerization function of the C-terminus of TRal (discussed below) include sequences common to the two proteins in addition to their unique regions. Despite their apparently different affinities for DNA, (~2, like TRal , forms two DNA-binding complexes with TREp. In the case of TRcul, methylation interference
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DNA Bindingby Thyroid Hormone Receptor and Variant
revealed that the upper complex is a dimer contacting both half-sites in TREp, whereas the lower monomeric complex contacted a single half-site (23). Unfortunately, it has been technically difficult to perform similar analysis of the tu2 complexes because the reduced number of DNA-protein complexes has resulted in an unacceptably low signal-to-noise ratio (not shown). It nevertheless seems likely that the two cu2 complexes represent monomer and dimer. However, no heterodimeric complexes were observed when a2 was mixed with T&l. It is possible that the TRal/a2 heterodimer forms but is unable to bind TREp and is therefore not detected in the band shift assay. This would be quite different from other DNA-binding proteins with related dominant negative partners where transcriptionally inactive heterodimers readily bind to DNA (44-47). Furthermore, if 012 forms non-DNA-binding heterodimers with TRal , the data suggest that this is a weak interaction which only occurs at very high concentrations of ot2. Another important difference between TRal and a2 is that c~2 does not heterodimerize with TR-binding proteins present in liver or in JEG3 (30) or COS-7 cells (data not shown). The failure of ot2 to heterodimerize with nuclear proteins which enhance DNA binding by TRs in vitro makes it highly unlikely that a2 inhibits T3 action by competing for these potential coregulators. Also, as discussed above, the a2/TRal interaction is weak at best, arguing against significant formation of potentially inactive heterodimers. Our data do not rule out the hypothesis that 012directly competes with TRs for TRE binding, although since the inhibitory effects of (~2 are relatively stoichiometric (35, 36), posttranslational modification of (~2 could be required to improve its DNA binding in vivo. It also remainspossiblethat a2 squelchestranscriptionalactivation by T3 by interacting with limiting transcription factors other than those whose binding to the TR is evident in the gel mobility shift assay. In addition to the functional implicationsof our findings, the inability of ot2 and a2AC to interact with TRbindingnuclearproteins highlightsthe active role of the C-terminusof TRotl , which contains only 40 aa which are not present in 012.This is confirmed by the finding that, like (~2,C-terminaldeletion mutants of the TR do not heterodimerize. The point at which TRcvl and cu2 diverge, illustrated in Fig. 6, has previously been identified (49) as highly conserved amongthe TR, RAR, and vitamin D receptors, all of which can heterodimerize with nuclear proteins (23, 27-31, 50-52). Particularly interestingis an Asp-Leu-Arg (DLR) motif (boxed in Fig. 6) which is common to the heterodimerizingreceptors but lacking in a2. This region is also the ninth of nine heptad repeatssuggestedby Formanand Samuels(53) to be involved in TR dimerization. While this paper was being revised, this region was also implicated in the heterodimerizationbetween the TR and retinoid X receptor-p (see Ref. 61). We initially hypothesized that the unique 40 aa of TRal might be sufficient for heterodimerization. How-
811
TRal RARa VDR c-erbAa2
MIGAC SISAK SLNEE SSILX
6. The Divergenceof c-erbAa2interruptsa Conserved Regionof a NuclearHormoneReceptorSubgroup Aminoacids366-380 of TRal and c-erbAa2are shown (seeFig. 1A). Humanandrat TRa!andp isoformsareidentical and RARPdiffersby a singleaminoacidfrom RARain this Fig.
region. The human RAR (58, 59) and vitamin D receptor (60) are shown, andthe number of the firstaminoacidisindicated. Homologous or identical sequences are shaded. The arrow points to beginning of divergence of ot2 (underlined italics).
ever, fusion of this sequence to the end of a2AC did not transfer the ability to interact, while addition of the final 100 aa of TRal did. Analysis of the data from all chimerasstudied suggeststhat the heterodimerization domain is contained within the final 70-100 aa. The fact that all additional aa except the last 40 are duplicated in the a2AC/TRal chimera suggests that, in addition to primary sequence information, the protein must be ableto assumea specificconformationin order to heterodimerize. Interestingly, the region lacking in the 70-aa chimera but present in the lOO-aa adduct, namely aa 31l-340 of TRal , overlaps an amphipathic a-helical sequence(288-331 of TRal) which was recently demonstratedto be important for interactionwith nuclear factors by Spanjaard et al. (54). Also of note, O’Donnell et a/. (55) have identified a highly conserved sequencecorrespondingto aa 236-255 of TRal [also referred to as the 7, domain by Forman and Samuels (53)], which is required for heterodimerizationof TRPl (30). Since 012,CU~AC,and a number of the TRal Cterminal mutants contain both the 236-255 and 288331 sequencesof TRal yet are unableto heterodimerize, we conclude that neither motif is sufficient. We proposethat at leasttwo, and possiblythree, interfaces are required for heterodimerization;the C-terminal100 aa of TRoll is sufficient for heterodimerization in the context of the ol2/TRal chimerasbecause it supplied one (31l-41 0) or two (31l-340 and 371-410) of the necessaryinterfaces,while the (Y~ACbackbone, which containsthe domaindescribed by O’Donnellet al. (55) provided the other. We are currently testing this model by attempting to transfer the ability to interact with TRbinding proteins to heterologous transcription factors with unrelated DNA-bindingmotifs.
MATERIALS
Synthesisof
AND METHODS TRal
and a2 in E. co/i
The rat TR4 and 012 cDNAs were subcloned into a pET3a vector (56) with 51 base pairs preceding an EcoRl cloning site such that the proteins were fused to the sequence MDYKEE-
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MOL 812
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Vol6
KARRASVQF (modified vector kindly provided by M. Blanar, UCSF, San Francisco, CA). The underlined amino acids are recognized by the FLAG M2 monoclonal antibody (IBI, New Haven, CT). An in-frame EcoRl site (encoding QF) was substituted for the 5’-untranslated region of both TRcul and a2 cDNAs using the polymerase chain reaction (PCR) with upstream primer: ~~GCCGGATCCGAATTCATGGAACAGAAGCCAAGC and downstream orimer TCCTCTCCCGGTTCTGC. The 3’ primer was located downstream of a unique Ncol site so that the altered 5’ portion could be inserted as a 375-base pair cassette into the cDNA, in order to reduce the possibility of introducing a mutation due to amplification. Sequences of the modified cDNAs were verified by direct sequencing using the dideoxynucleotide method and Sequenase (US Biochemicals, Cleveland, OH) as well as by restriction analysis (58). Expression plasmids were grdwn in the BL2l(pLYSs) strain of E. co/i and induced with isopropyl-p-othiogalactopyranoside. Soluble protein preparations were made by resuspending pellets from 500 ml bacterial (A, 0.30.5) in 5 ml 50 mM Tris DH 8. 50 mM NaCI. 1 mM EDTA. and 0.2’ mM phenylmethylsuifonyl fluoride. The cells were lysed with lysozyme (2 mg/ml final), and incubated at 0 C for 30 min, after which 50 ~1 of a solution containing 0.5% sodium deoxycholate and 10% Triton X-l 00 were added and incubated an additional 20 min. Next, the suspension was sonicated at 0 C, then centrifuged to remove insoluble material. Western analysis of the supernatant and insoluble fractions indicated that approximately 5-20% of the recombinant protein was soluble, depending on the preparation. Over-sonication increased solubility at the expense of DNA-binding activity. The TRotl and c-erbAa2 preparations were usually made simultaneously, and at least three independent preparations of TRnl and c-erbAa2 gave similar results. The glutathione-S-transferase-TRal (123-410) fusion protein was prepared by inserting a PCRderived fragment encoding amino acids 123-410 of TRal in frame into the BamHl site of pGEX-PT (Pharmacia, Piscataway, NJ). Prepartation
of Monoclonal
Antibody
to T&l
TRal was synthesized in E. co/i as a fusion protein with six histidines at the amino terminus (62). The protein was purified to apparent homogeneity on a nickel affinity column (62) cut out of a preparative polyacrylamide gel, and used to immunize mice. Hybridoma cell lines were screened by enzyme-linked immunosorbent assay, and an immunoglobulin M (IgM) antibody which recognized TRoll was purified by affinity chromatography. Western
Analysis
Extracts from bacteria expressing TRal and c-erbAa2 were electrophoresed along with control extract in 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to nitrocellulose for 16 h overnight in 25 mM Tris-HCI, 192 mM glycine, and 20% methanol, after which the blots were blocked for 1 h with 5% powdered milk in lx PBS, 0.2% Tween-20 (57), and washed three times for 5 min in Buffer A (1 x PBS, 0.05% Tween-20). Blots were incubated for 2 h with antiFLAG mouse monoclonal antibody M5 (Immunex/lBI) or purified TRa monoclonal antibody diluted l:lO,OOO in lx PBS, 1% milk, and 0.05% Tween-20 (Buffer B), washed three times for 5 min with Buffer A, incubated with goat antimouse IgG (FLAG) or goat antimouse IgM (TR moniclonal) coupled-to horseradish peroxidase diluted 1 :lOO,OOO in Buffer B. washed three times ‘for 5 min with Buffer A, and developed using chemiluminescent detection (ECL; Amersham, Arlington Heights, IL). Synthesis
of Proteins
in Reticulocyte
Lysates
Wild type TRnl or c~2 cDNAs in pBluescript (PBS) were linearized with an appropriate restriction enzyme before in
No. 5
vitro transcription with T3 or T7 RNA polymerase. RNA synthesized in this way was translated in rabbit reticulocyte lysste (Promeqa, Madison, WI) in the presence of 135Slmethionine. Protein-synthesis was quantitated by a comdinaion of autoradiography and trichloroacetic acid (TCA) precipitation, taking variations in methionine content into account. Of all the proteins described in this report, only wild type TRotl specifically bound T3 (data not shown). Generation
of TR and 02 Mutants
and Chimeras
Deletion mutants were made by cutting TRotl or 012 cDNAs in PBS with restriction enzymes that cut within the coding sequences before in vitro transcription/translation. The 012Ac mutant was made by either cutting with Sacl or by using PCR to introduce a stop codon after the Sacl site (this construct is referred to as cu2AC-.Sac/stop). The 3’ primer (GGGGCGGCCGCTCACAGGGAGCTTGCCTCACTG) for this reaction is complementary to a2 through codon 470 and is followed by a stop codon and a NotI site; the 5’ primer (TGACAGCTGCTGCGTCATCG) is complementary to r~2 upstream of the unique Ncol site in the cDNA. PCR with these primers and subsequent digestion with Not1 and Ncol produced a truncated c~2 fragment that replaced the 3’ end of the ot2 cDNA in PBS. In vitro transcription from this template and translation of the resulting RNA in rabbit reticulocyte lysate (Promega) produced a protein which was indistinguishable from a2 truncated by restriction enzyme cleavage and translated from runoff transcription of the template both by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and gel shift assay (data not shown). The cu2ACSac/stop construct was used in the generation of the n2/al chimeric proteins. For these constructs, the Cal cDNA was amplified with the Ml3 universal primer 3’ to the cDNA in PBS and a series of 5’ primers, each consisting of a Sacl site in frame with TRcvl-specific sequences. Amplified fragments contained 40, 70, 100, and 150 aa from the Cterminus of TRal (using the primers GGGGAGCTCCGTGACTGACCTCCGCATGAT,GGGGAGCTCCAAGAGTCAGGAGGCCTACCT,AAAGAGCTCCCTGGATGATACGGAAGTGGC. and AAAGAGCTCCCTGCGGGCAGCTGTCCGCTA. respectively) were subcloned in-frame into theSac site of n2& Sac/stop. Each construct produced a protein of the appropriate size (see Results). Electrophoretic
Mobility
Shift Assays
Gel shift assays were performed as previously described (23). When labeled DNA was used, as in most experiments, DNA concentration was approximately 0.5 rig/binding reaction. When unlabeled DNA was used in order to track labeled proteins directly, 250 ng DNA were present in each reaction. Liver nuclear extracts were prepared as previously described (23), and 2-5 pg extract were used per reaction as indicated. Also, FLAG antibody was added as indicated in some cases.
Acknowledgments We thank Dr. Ron Koenig (University of Michigan, Ann Arbor, Ml) for confirminq the estimate of active TRal orotein svnthesized in E. co/i. we also thank Dr. Michael Blanar (Uniiersity of California, San Francisco) for providing the FLAG-containing bacterial expression vector, and Frank Rauscher for providing the (His)G-expression vector.
Received January 3, 1992. Revision received March 3, 1992. Accepted March 10, 1992. Address requests for reprints to: Dr. Mitchell A. Lazar, University of Pennsylvania, CRB 611, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104-6149. This work was supported in part by an NSF predoctoral
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DNA Binding
by Thyroid
Hormone
Receptor
and Variant
training grant (to D.K.), by NIH Grant 1 ROl DK-43806-01 (to M.A.L.), and by an American Federation for Clinical Research Foundation-Merck Early Career Development Award (to M.A.L.).
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