Thyroid Hormone Receptor Dimerization Function Maps to a Conserved Subregion of the Ligand Binding Domain

Jae Woon Lee, Tod Gulick,

and David D. Moore

Department of Molecular Biology Massachusetts General Hospital Boston, Massachusetts 02114

Thyroid hormone receptors (TRs) bind as dimers to specific DNA response elements. We have used a genetic approach to identify amino acid sequences required for dimerization of the TRP isoform. Bacteria expressing a chimeric repressor composed of the DNA binding domain of the bacteriophage X cl repressor fused to the TRP ligand binding domain are immune to X infection as a consequence of homodimerization activity provided by the receptor sequences. The phenotypes of deletions and point mutations of the TRP sequences map dimerization activity to a subregion of the ligand binding domain that is highly conserved among all members of the nuclear hormone receptor superfamily. These results confirm and extend previous findings indicating that this subregion plays an important role in the dimerization of TR@ and other superfamily members. (Molecular Endocrinology 6: 1867-1873, 1992)

Previous studies have mapped dimerization activity to various domains of the conserved structure shared by superfamily members. In the case of the glucocorticoid receptor, direct structural analysis has demonstrated a homodimerization interface within the DNA binding or C domain (15). Sequences just distal to the DNA binding domain (16) and in the C-terminal part of the ligand binding (E) domain (17, 18) have been implicated in homodimerization of the estrogen receptor. The analogous subregion of the E domain has been proposed to be involved in heterodimerization of both TR and the retinoic acid receptors (RARs) (19-21). This part of the E domain includes heptad repeats of hydrophobic residues that have been proposed to form a leucine zipper dimerization interface (22). However, interpretation of many of the studies with TR is complicated by the fact that dimerization activity is associated with receptor domains where nuclear localization, transcriptional activation, and ligand binding activities reside, and methodologies used depend on these properties for scoring dimerization. We have used a recently developed genetic approach to the analysis of protein-protein interactions (23) to map amino acid sequences required for homodimerization of the p-isoform of rat TR (rTR). This system, based on genetic screening of the function of bacteriophage X cl repressor fusion proteins in Escherichia co/i, permits segregation of dimerization from transcriptional regulation and other activities. The cl repressor consists of an amino-terminal DNA binding domain (DBD) and a functionally and structurally distinct dimerization domain. The repressor acts to shut off viral gene expression by binding to specific operator sites in essential regulatory regions of the viral genome, rendering bacteria expressing intact cl immune to viral infection (24). Since dimerization of the repressor is essential for efficient DNA binding, expression of the cl DBD alone is not sufficient to confer immunity. However, repressor function can be rescued by fusion with a heterologous dimerization domain (23). Here we describe the activity of chimeric repressors in which dimerization function is provided by the ligand binding domain of TRB.

INTRODUCTION The nuclear hormone receptor superfamily is a group of proteins linked by conserved structure and function that includes the receptors for a variety of small hydrophobic compounds such as steroids, thyroid hormone (T3), and retinoids. All of the members of the superfamily examined to date function as transcriptional regulators by binding to DNA as dimers (reviewed in Refs. l3). While the steroid receptors bind as homodimers to simple palindromic response elements, head-to-head or tail-to-tail inverted repeats and direct repeats of a hexameric consensus can function as T3 response elements. T3 receptors (TRs) can bind to such sites as homodimers (4-7) and the binding affinity of the homodimers to a wide variety of wild type and mutant elements correlates strongly with their response to T3 (4). TRs can also bind with even higher affinity to the same array of sites as heterodimers with the retinoid X receptors (RXRs; 8-l 4). 0888-8809/92/1867-1873$03.00/0 Molecular Endocrmology CopyrIght 0 1992 by The Endocrine

Society

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RESULTS A Chimeric Repressor Including the Hinge and Ligand Binding Domains of Rat TR@ Confers Immunity to X Infection

A DBD TRP

In the vector pYCl84cl, expression of the bacteriophage Xcl repressorDBD (aminoacids l-l 12) is under the control of an isopropylthiogalactoside(IPTG)-inducible promoter. A fragment of the rTR/I (25) encompassing the C-terminalend of the DBD and the entire hinge and ligand binding domains(D, E, and F, amino acids 169-461) was inserted into this vector to generate pYCcl/TRP (Fig. IA). As shown in Fig. lB, E. co/i transformed with pYCcl/TRP are immuneto high titer Xinfection when expressionof the chimericgene product isinduced(+IPTG), but not inthe absenceof inducer (-IPTG). The presenceor absenceof T3 has no effect on the immunityphenotype of the chimera,as expected from previous findingsthat the hormoneis not required for T3 receptors to interact with specific DNA binding sites in viva (26-28) or in vitro (4, 29, 30). Bacteria transformedwith pYCcl are susceptibleto X infection even under inducing conditions, and cells containing either plasmidare susceptibleto infection by a hybrid strain of X with an immunity region derived from 480, a related bacteriophagethat is not subject to repression by X cl. Thus, the chimericrepressor acts like the wild type cl protein in conferring specific immunity to X infection, demonstratingthat the hingeand ligandbinding domainsof TRPcontain a homodimerizationactivity. The chimeric repressor expressed from pYCcl/TR@ bindsT3 specificallyand is presentat low concentration in the immunecells. Crude extracts of cells grown in the presenceof IPTG were incubated with [‘251]T3in the presenceor absenceof an excess of unlabeledT3 and subjectedto molecularsieve chromatography.The amount of T3 specificallybound at saturation indicates that approximately 50 chimera monomersare present per cell, a concentration similarto that of the cl repressor in a normallysogen(24). This low levelof expression is a reflection of the low copy number of the pACYC vector and the weaknessof its P-lactamasepromoter, both of which are componentsof a strategy designed to maximizethe sensitivity of the assay. Specific ligand bindingwas taken to indicate proper protein folding of the E domain of the chimera, as expected from the previously describednormalligandbindingaffinity of E. co/i-expressednative TRP(31). The apparent mol wt of the specific T3 complex indicated the formation of dimersor higherorder oligomersof the chimericprotein. This is consistent with a recent report that a purified fragment of the rTRcv1consistingonly of the E domain can form dimersin solution(32). TRj3 Homodimerization Function Maps to a Conserved Portion of the Ligand Binding Domain

HEPTAD

El

3

1 A/B

D

C

E

F

X DBD

DIMER

CI

1

km m

cI/TRP

6

pYCclTRP

pYCl84cl

-IPTG

+IPTG

0 pfu:

lrl” 1: lo”

480 h

000 000

Fig. 1. The E Domain of TF@ Restores X Immunity Function to The X Repressor DBD A, The primary structures of wild type TR@, X cl repressor and a chimeric repressor protein are depicted. TRP consists of the N-terminal A/B domain, the DBD, the hinge or D domain, the E domain, and the C-terminal F domain. The positions of the conserved El subdomain and the heptad repeats of hydrophobic residues (19, 22) are indicated. The repressor consists of an N-terminal DBD and a C-terminal dimerization domain (DIMER). B, Freshly poured lawns of E. co/i (strain XL1 B) transformed with plasmids expressing either the DBD of the cl repressor (pYC184cl) or the chimeric repressor (pYCclTRP) were infected by spotting with 100-111 aliquots of dilutions of lysates of the X mutant KH54 (X) or Charon (480). The titer and pattern of the aliquots is indicated. The absence or presence of the inducer IPTG is indicated.

The immunity phenotype of deletion mutants was used to identify regionsof the ligandbindingdomainessential The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 10 August 2016. at 07:29 For personal use only. No other uses without permission. . All rights reserved.

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for dimerization activity. Mutants with short deletions extending into the hinge (D) domain (Fig. 2, Dl and D2) maintain immunity function, consistent with studies demonstrating that this region is not necessary for function of steroid receptors (33,34). However, repressor function was also observed in several additional chimeras retaining only the proximal portion of the D domain. This finding is in agreement with previous results mapping an independent but subsidiary dimerization function to this region of the estrogen receptor (16) and TRa (6). This region was not included in the further series of E domain deletion and point mutants. A number of chimeras with internal or C-terminal deletions in the region of the E domain proposed to form a leucine zipper-like dimerization interface are functional repressors (mutants D4-9 and 011). The phenotypes of these deletion mutants rule out a primary role for this region in homodimerization. This is consistent with some recent results with TRa (6), and with the fact that the primary structure of this region is not generally consistent with a coiled coil/leucine zipper. However, results reported for the estrogen receptor (17, 18) support some role in homodimerization for a conserved subregion at the C-terminal portion of the E domain. This subregion has also been implicated in TR (19-21) and RXR (9, 10, 13) heterodimerization. In these studies the activity of this subregion was only examined in the presence of other receptor dimerization domains, and our results indicate that it is neither necessary nor sufficient for homodimerization of the chimeric repressor. Together, the N- and C-terminal deletions map a region essential for TRP homodimerization to amino

acids 283-313. This interval consists largely of the subregion called El (35), see Fig. 4A), the most strongly conserved portion of the E domain. Dimerization function is eliminated in a C-terminal truncation that removes part of El (D12) and by two small deletion mutations that lie entirely within this subregion (D13 and 14). To exclude the possibility that the phenotype of these mutants results from an indirect alteration in the structure of the E domain, rather than a specific effect on dimerization, we introduced a series of previously studied amino acid substitutions into the El subdomain of the cl/TRP chimera. These mutations have been shown to inactivate the transcriptional regulatory function of the receptor in cotransfections and to prevent interaction with the T3 receptor accessory protein (TRAP) (36, 37). The alterations of TRP amino acids 288, 290, and 300 were of particular interest, since these mutant TRs have been shown to bind T3 with normal affinity and could, therefore, be confidently assumed to have an authentic tertiary structure. As shown in Fig. 2, two of these three substitutions (mutants L29OS and D300A) inactivate function of the fusion protein. The Ala to Asp substitution at TRP residue 287 that inactivates ligand binding in mutant receptors also inactivates immunity function when introduced into the chimera. To directly verify these in vivo findings and to exclude attribution of the immunity phenotype to potential differences in the concentration of mutant chimeras in bacteria, we examined the effect of El point mutations in a cell-free system. Full-length wild type and mutant fusion proteins were produced by in vitro transcription and translation, and their dimerization function was examined in gel mobility shift assays (Fig. 3). As ex-

#

F ..( w ~-----’ v, + ) e’ k-----.’ +....., w ......-(~ F , b-----F ......-( -*...m w +--* F-~---~ ~-~----’ ~-~~---~

-

HEPTAD

El

mx

8 , I , I I I

,,,,,/-,,>”

---

....----.---zx,. >-----.-----..mz”

,................

-

(=1

zzzz2m

.

........

“,,,z~z,A v-n,,-

J ,

l . l

r------

-

WT (169-461) Dl D2 D3 D4 D5 D6 D7 D8 D9 DlO Dll 012 013 D14 Pl P2 P3 P4

MUTATED AMINO ACIDS

d169-182 d169-199 d169-282 d333-343 d314-356 d321-360 d379-423 d379-440 d359-442 d281-428 d378-461 d292-461 d286-293 d287-305 A287D K2881 L29OS D300A

h IMMUNITY ++ -I-+ ++ + ++ + + ++ ++ +

++ ++ -

Fig. 2. TR$ Homodimerization Activity Maps to the Conserved

El Subdomain The indicated deletion mutations were introduced into the cl/TR@ chimera. Deleted amino acids in the rTRp sequence. Amino acids substituted in each mutant are as noted: the wild type amino position and the substituted amino acid, e.g. in A287D an alanine at position 287 is replaced by an immunity to high titer X infection as shown in Fig. 1; +, immunity to infection by titers 1 OO-fold plaques on a sensitive host; -, full sensitivity to infection.

are listed according to position acid is listed, followed by the aspartic acid (36, 37). ++, Full higher than necessary to yield

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cI/TR CT DBD L29OS D3OOA operator

NS

Vo16No.11

+

+

+

+

+

+ +

+

+ + + + + +

+ + + + + + \\

.\

Fig. 3. Mobility Shift Assays Confirm Genetic Mapping of Dimerization Activity to El The wild type chimeric cl/TRP (cl/TR), X repressor DBD alone (cl DBD), and two substitution mutant chimeric repressors (L29OS and D300A) were produced by in vitro transcription and translation, and equivalent amounts of the proteins were used for gel shift analysis. Binding reactions included the indicated proteins with no competitor (lanes l-4) a 40-fold excess of a nonspecific competitor oligonucleotide (NS), or a 40-fold excess of the unlabeled operator oligonucleotide.

petted from previous results demonstrating that dimerization is essential for high affinity DNA binding by the repressor, the cl N-terminal DBD alone showed no binding to a symmetric X operator site. However, substantial binding was observed with the wild type cl/TR chimera, demonstrating that the receptor sequences can confer dimerization function. In contrast, binding of equivalent amounts of either of the nonfunctional substitution mutant proteins (L29OS and D300A) was dramatically reduced. These data directly confirm that the wild type TRP chimera forms dimers in vitro substantially more effectively than the El point mutants. We conclude that immunity phenotype is an accurate assay of homodimerization and that the conserved El subregion has a direct role in dimerization of TRP.

DISCUSSION The results described here demonstrate that the E domain of the rTF@ isoform contains a dimerization activity that can restore repressor function to the Nterminal DBD of the bacteriophage X cl protein. The highly conserved El subregion is the only portion of the TR E domain that is essential for this dimerization function. Since all of the members of the nuclear receptor superfamily examined to date bind as dimers to their response elements, we suggest that the strong con-

servation of the amino acid sequence of the El subdomain throughout the superfamily may be a reflection of a conserved role for this element in dimerization. This subregion has been associated with other functions, particularly the interactions of the glucocotticoid and estrogen receptors with HSPSO (38-40). Since the TRs (41), and perhaps other members of the superfamily (42) do not interact with HSPSO, this function is not likely to be the basis for the conservation of this subregion. Although the results described here are based on analysis of TR homodimerization, they may also be relevant to heterodimerization. General support for this possibility is found in the strong similarity in DNA binding specificity of TR homodimers and TR/RXR heterodimers (8-13) which suggests that the protein-protein contacts of the two complexes may be very similar. More specific support is provided by the effects of El mutations on interaction of TRP with TRAP, a protein activity that stimulates binding of TR to T3 response elements (43,44), apparently by formation of TR/TRAP heterodimers (5, 45). The two TRP substitution mutations within El that prevent homodimerization of the chimeric repressor also strongly decrease interaction of mutant receptors with TRAP in vitro and decrease function of the receptor in cotransfections (36, 37). Moreover, the substitution at amino acid 288 that does not prevent homodimerization of the chimeric repressor has a significantly weaker effect on the in vitro interaction with TRAP (37). Recent results demonstrate that the TRAP effects can be explained by the activities of the RXRs, which form strong heterodimers with the TRs and with other receptors. Thus, the precise overlap between the effects of the El mutations on activity of the repressor chimeras and on TRAP interactions suggests that El is similarly involved in homodimerization of TRP and in its heterodimerization with RXRs. Further evidence for a conserved role for the El segment in heterodimerization is provided by the fact that deletion of that subregion of RXRP prevents heterodimerization with TR (13). Strong additional support is provided by a recent preliminary report that introduction of mutations similar to those described here into the vitamin D receptor (VDR) and RARa proteins strongly decreases their function in cotransfections and their ability to cross-link to RXRP or to a 63-kilodalton protein with TRAP activity (46; Rosen, E., and R. Koenig, personal communication). Thus, although recent biochemical evidence supports a primary role for TR/ RXR heterodimers rather than homodimers (30) we conclude that the analysis of homodimerization by the approach described here can provide important information on the protein-protein interactions of TRP. El sequence similarity is high among the members of the family (T3, retinoic acid, vitamin D, and 9-&sretinoic acid receptors and the orphan superfamily member COUP-TF, chicken ovalbumin upstream promoter transcription factor) reported to form heterodimers (8-l 3, 19, 20, 22, 47). This region is also strongly conserved among the steroid receptors, all of which

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Dimerization

apparently bind DNA only as homodimers. In several positions (shaded in Fig. 4A), the particular amino acid conserved in one of the two groups is different from that conserved in the other group. These positions may contribute to the specificity of the dimeric interactions of each group. Although additional sequences in the E domain are not required for TR homodimerization in the results described here, the conserved E3 subregion located near the C terminus of the E domain (35) appears to be essential for heterodimerization of TRs, RARs, and RXRs (10, 13, 20, 21). This short subregion, which includes heptad 9 of Forman and Samuels (22) is also required for homodimerization of the estrogen receptor

(17, 18). It is interesting that there is some direct amino acid sequence similarity between the E3 subdomain and a portion of the El subdomain in the group of proteins that form heterodimers. The VDR shows the best match, with 6 of 11 identical residues plus a single conservative substitution, while TR@ is more typical, with 3 of 11 identities and two conservative substitutions (Fig. 4B). Further analysis will be necessary to determine whether the El and E3 subdomains have coordinate functions in dimeric interactions. The genetic system described here may be useful in defining the specificity in these interactions.

MATERIALS

RARa VDR RXRa COUP

** * * VDFAKKLPMFCELPCEDQIILLKG VEFAKOLPGFTTLTIADOITLLKA IGFAKIiIPGFRDLTSED~IVLLKS VEWAKRIPHFSELPLDDQVILLRA VEWARNIPFFPDLQITDQVSLLRL

GR MR PR AR

VKWAKAIPGFRNLHLDDQMTLLQY VKWAKVLPGFKNLPLEDOITLIOY VKWSKSLPGFRNLHIDD&TLI;Y VKWAKALPGFRNLHVDDQMAVIQY

TRP

I

III

II

I

I

III1

II

B.

pYCl84cl is a derivative of pACYC177 (48) in which expression of the N-terminal DBD (amino acids l-l 12) of X cl is controlled by a derivative of the B-lactamase promoter containing a lactose operator (kindly provided by C. Bunker and R. Kingston, Department of Molecular Biology, Massachusetts General Hospital). pACYCl77 contains the origin of replication from pl5A and is a low copy replicon compatible with plasmids like pBR322 and pUC19 that contain the colE1 origin. pYCcl/ TR@ was constructed by insertion of a fragment from the rat TRB coding region encompassing amino acids 169 to the Cterminus (461) into this vector. Deletions and previously described amino acid substitutions (36, 37) were introduced into pYCcl/TRp using convenient restriction sites and standard methods (49). In some cases, Bal31 exonuclease was used to create a random set of deletions from a single site. Both substitution and deletion mutations were verified by direct DNA sequencing. For DNA binding studies, repressor coding regions were subcloned into a derivative of pBluescriptSK that contains a mammalian consensus sequence for efficient translation initiation. X Immunity

TRP

FAKKLPMFCELP WPKLLMKVTDLR

El E3

VDR

FAKMIPGRFDLT YAKMIQKLADLR

El E3

Fig. 4. El and E3 Sequences A, Conservation of the El subdomain among superfamily members. The top group includes El sequences from rTR@ and RAFta, VDR, and RXRa, all of which have been reported to form heterodimers with TR, as well as COUP-TF, an orphan member of the superfamily reported to form heterodimers with RXR. TRP sequence is from amino acids 284-306 (25). Positions altered by substitution mutations are indicated by asterisks. The bottom group includes four steroid receptors, all thought to form only homodimers. GR, Glucocorticoid receptor; MR, mineralocorticoid receptor; PR progesterone receptor; AR androgen receptor. Except for rTRB, human cDNA sequences are indicated (see Ref. 53 and refs. therein). Positions conserved in both groups are indicated by bars. Positions that show conservation of different amino acids in the two groups are stippled. B, Similarity of El and E3 sequences from TRB and VDR. The entire E3 sequence and only a portion of the El sequence is included. Identical or conserved amino acids are shaded.

AND METHODS

Assay

Lawns of E. co/i strains containing various plasmids were infected by spotting with O.l-ml aliquots of serial dilutions of bacteriophage X lysates containing from 1 02-1 0’ plaque forming units. X Strains included X KH54, which has X host range and immunity, and Charon3, which has X host range but an immunity region from the related bacteriophage $80 and is not sensitive to X repressor (50). Infected lawns were incubated at 37 C for 8-l 8 h. DNA Binding

Assay

Proteins were produced in vitro using bacteriophage T7 RNA polymerase and rabbit reticulocyte lysates as described (51). Equivalent amounts of full-length versions of each, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography of %-labeled products, were used in DNA binding reactions. Binding reactions included 0.2 fmol of each protein and 1.5 pmol of a double-stranded 32Plabeled oligonucleotide probe containinq a consensus h-operator site (5’-AATTCCACATGCAACCATTATCACCGCCG’GTGATAATAGTCGG-3’) in a buffer consistina of 25 mM Tris IDH 7.5) 50 mM KCI, 10% glycerol, 6 mM Ca&, 1.5 mM MgCI,, 0.3 mM EDTA, 0.5 mM dithiothreitoi, 50 Kg/ml BSA, and 50 @g/ml polydl.dC) (52). In some reactions, a 40-fold excess of unlabeled X-operator oligonucleotide or an unrelated doublestranded oligonucleotide were added as competitor. Free and protein-bound DNA were resolved by electrophoresis on a 5%

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polyacrylamide (49).

gel in 0.5~

Tris-buffered

EDTA

as described

14.

Acknowledgments We thank Chris Bunker, Bob Kingston, Jim Hu, and Ron Koenig for helpful discussions, Chris Bunker and Bob Kingston for pYCl84cl, and Ron Koenig for the mutations affecting El.

15.

16. Received July 9, 1992. Revision received August 18, 1992. Accepted August 18, 1992. Address requests for reprints to: Dr. David Moore, Department of Molecular Bioloav. Massachusetts General Hospital, Boston, Massachusetts ?I21 14. This work was supported by PHS Grant DK-43382 from the NIDDK and by a grant from Hoechst AG.

17.

ia.

19.

REFERENCES 1. Brent GA, Moore DD, Larsen PR 1991 Thyroid hormone regulation of gene expression. Annu Rev Physiol 53:1735 2. Beato M 1969 Gene regulation by steroid hormones. Cell 561335344 3. Evans RM 1966 The steroid and thyroid receptor superfamily. Science 240:889-a95 4. Williams GR, Harney JW, Forman BM, Samuels HH, Brent GA 1991 Oligomeric binding of T3 receptor is required for maximal T3 response. J Biol Chem 266:19636-19644 5. Lazar MA, Berrodin TJ, Harding HP 1991 Differential DNA binding by monomeric, homodimeric and potential heteromeric forms of the thyroid hormone receptor. Mol Cell Biol 11:5005-5015 6. Zhang X-K, Tran PB-V, Pfahl M 1991 DNA binding and dimerization determinants for thyroid hormone receptor N and its interaction with a nuclear protein. Mol Endocrinol 5:1909-1920 7. Holloway JM, Glass CK, Adler S, Nelson CA, Rosenfeld MG 1990 The C-terminal interaction domain of the thyroid hormone receptor confers the ability of the DNA site to dictate positive or negative transcriptional activity. Proc Natl Acad Sci USA 87:8160-8164 a. Yu VC, Delsert C, Anderson B, Holloway JM, Devary OV, Naar AM, Kim SY, Boutin J-M, Glass CK, Rosenfeld MG 1991 RXR& a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements. Cell 67:1251-l 266 9. Zhang X-K, Hoffmann B, Tran PB-V, Graupner G, Pfahl M 1992 Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors. Nature 355:441446 10. Lied M, Kastner P, Lyons R, Nakshatri H, Saunders M, Zacharewski T, Chen J-Y, Staub A, Garnier J-M, Mader S, Chambon P 1992 Purification, cloning and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently. Cell 68:377395 11. Kliewer SA, Umesono K, Mangelsdorf DJ, Evans RM 1992 Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling. Nature 355:446-449 12. Bugge TH, Pohl J, Lonnoy 0, Stunnenberg HG 1992 RXRtu, a promiscuous partner of retinoic acid and thyroid hormone receptors. EMBO J 11 :1409-i 418 13. Marks MS, Hallenbeck PL, Nagata T, Segars JH, Appella E, Nikodem VM, Ozato K 1992 H-2RIIBP (RXR) heterodimerization provides a mechanism for combinatorial diversity in the regulation of retinoic acid and thyroid hormone responsive genes. EMBO J 11 :1419-l 435

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11

Hallenbeck PL, Marks MS, Lippoldt RE, Ozato K, Nikodem VM 1992 Heterodimerization of thvroid hormone (TH) receptor with H-2RIIBP (RXRB) enhances DNA binding and TH-dependent transcriptional activation. Proc Natl Acad Sci USA 69:5572-5576 Luisi BF, Xu W-X, Otwinowski Z, Freedman LP, Yamamoto KR, Sigler PB 1991 Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature 352:497-505 Kumar V, Chambon P 1968 The estrogen receptor binds tightly to its responsive element as a ligand induced homodimer. Cell 55:145-l 56 Fawell SE, Lees JA, White R, Parker MG 1990 Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor. Cell 60:953-962 Lees JA, Fawell SE, White R, Parker MG 1990 A 22 amino acid peptide restores DNA binding activity to dimerization defective mutants of the estrogen receptor. Mol Cell Biol 10:5529-5531 Forman BM, Yang C-R, Au M, Casanova J, Ghysdael J, Samuels HH 1967 A domain containing a leucine zipper like motif mediates novel in viva interactions between the thyroid hormone and retinoic acid receptors. Mol Endocrinol 3:161 O-l 626 Glass CK, Lipkin SM, Devary OV, Rosenfeld MG 1969 Positive and negative regulation of gene transcription by a retinoic acid thyroid hormone receptor heterodimer. Cell 59:697-708 Selmi S, Samuels HH 1991 Thyroid hormone receptor and v-erbA. J Biol Chem 266:11569-l 1593 Forman BM. Samuels HH 1990 Minireview: interactions among a subfamily of nuclear hormone receptors: the regulatory zipper hypothesis. Mol Endocrinol 4:12931302 Hu JC, O’Shea EK, Kim PS, Sauer RT 1990 Sequence requirements for coiled-coils: analysis with X-repressorGCN4 leucine zipper fusions. Science 250:1400-1403 Ptashne M 1966 A Genetic Switch: Gene Control and Phage X Cell. Blackwell, Cambridge Koenia RJ. Warne RL. Brent GA. Harnev JW. Larsen PR. Moore DD 1968 Isolation of a cDNA clone’ encoding a biologically active thyroid hormone receptor. Proc Natl Acad Sci USA 65:5031-5035 Brent GA, Dunn MK, Harney JW, Gulick T, Larsen PR, Moore DD 1969 Thyroid hormone aporeceptor represses T3 inducible promoters and blocks activity of retinoic acid receptor. New Biologist 1:329-336 Damm K, Thompson CC, Evans RM 1969 Protein encoded by v-erbA functions as a thyroid hormone receptor antagonist. Nature 339:593-597 Graupner G, Wills KN, Tzukerman M, Zhang X-K, Pfahl M 1969 Dual regulatory role for thyroid hormone receptors allows control of retinoic acid receptor activity. Nature 340:653-656 Lavin TN, Baxter JD, Horita S 1968 The thyroid hormone receptor binds to multiple domains of the rat growth hormone 5’-flanking sequence. J Biol Chem 263:94169426 Yen PM, Darling DS, Carter RL, Forgione M, Umeda P, Chin WW 1992 Triiodothyronine (T3) decreases binding to DNA by T3-receptor homodimers but not receptorauxiliary protein heterodimers. J Biol Chem 267:35653568 Lin K-H, Fukuda T, Cheng S-Y 1990 Hormone and DNA binding activity of a purified human thyroid hormone nuclear receptor expressed in E. co/i. J Biol Chem 265:5161-5165 Apriletti JW, McGrath ME, West BL, Fletterick RJ, Baxter JD, Large scale expression of rat (~1 thyroid hormone receptor ligand binding domain in E. co/i and initial x-ray crystallography studies. Program of the 74th Annual Meeting of The Endocrine Society, San Antonio TX, 1992, p 444 (Abstract)

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