THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 16, Issue of June 5, pp. 11534-11541,1993 Printed in U. S. A.

Dimerization Interfaces of Thyroid Hormone, Retinoic Acid, Vitamin D, and Retinoid X Receptors*

(Received for publication, February 8, 1993)

Evan D. Rosen, Eric G. Beninghof, and Ronald J. Koenig From the Endocrinology Division, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0678

A subclass of erbA-related nuclear receptors has been shown to require interaction with an auxiliary protein(@ from nuclear extract in order to achieve high affinity DNA binding in vitro. The retinoid X receptor recently has been demonstrated to be such an auxiliary protein as it enhances specific DNA binding by thyroid hormone receptors, retinoic acid receptors, and the vitamin D receptor. Mutation of a highly conserved 20-amino acid region within the ligand-binding do- main of thyroid hormone receptor B disrupts its phys- ical association with auxiliary protein from JEG-3 cells as well as with recombinant retinoid X receptor B. The homologous 20-amino acid regions from retinoic acid receptor a and the vitamin D receptor also are critical determinants of the heterodimeric interaction between these receptors and JEG-3 cell auxiliary pro- tein as well as retinoid X receptor B. However, the same region of retinoid X receptor B appears to play a minor, if any, role in heterodimerization. In addition, transfection studies indicate that disruption of heter- odimerization impairs the ability of these receptors to function as ligand-dependent transcriptional activa- tors.

The erbA superfamily of ligand-inducible transcriptional activators includes the receptors for steroid and thyroid hor- mones (3,5,3’-triiodothyronine (T3)l), retinoic acid (RA), vi- tamin D, and a large group of so-called orphan receptors with unknown ligands and physiological roles (1). A specific subgroup of this superfamily has been shown to require the presence of a nuclear auxiliary protein in order to achieve high affinity DNA binding to specific response elements in vitro (2-7). We have designated this activity TRAP, for T3 receptor auxiliary protein. Other erbA receptors that interact with TRAP or TRAP-like activities are the retinoic acid receptors (RARs) and the vitamin D receptor (VDR) (8-10). All of these nuclear receptors bind to versions of the consensus hexamer AGGTCA, organized into combinations of direct and/or inverted repeat motifs (11-13).

The study of nuclear auxiliary proteins that associate with

* This work was supported by National Institutes of Health Grant DK44195. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: TB, 3,5,3’-triiodothyronine (thyroid hormone); CAT, chloramphenicol acetyltransferase; EMSA, electro- phoretic mobility shift assay; GH, growth hormone; PAGE, polyacryl- amide gel electrophoresis; RA, retinoic acid RAR, RA receptor; RARE, RA response element; RXR, retinoid X receptor; TR, T3 receptor; TRAP, TR auxiliary protein; TRE, T a response element; VDR, vitamin D receptor; VDRE, vitamin D response element; la,25- (OH)2D3, la,25-dihydroxyvitamin D3; bp, base pair($.

erbA superfamily members was advanced by the recent reali- zation that the retinoid X receptor (RXR) can substitute for TRAP activity from crude nuclear extracts (14-19). Three RXR genes have been identified, all of which encode erbA- related proteins that themselves are transcriptional activators in the presence of the ligand 9-cis-retinoic acid and an appro- priate response element (20-22). However, in addition to and separate from the above activity, RXR proteins have the ability to enhance specific DNA binding by erbA superfamily members. Thus, RXRs have been shown to increase the affinity of T3 receptors (TRs) for T3 response elements (TREs), RARs for RA response elements (RAREs), and VDRs for vitamin D response elements (VDREs), presumably by interacting with these receptors as heterodimers on the re- sponse elements. Additionally, RXRs have been shown to increase the transcriptional response to ligand when cotrans- fected with TRs, RARs, and VDRs in the presence of an appropriate response element (15, 17-19, 23).

Despite the obvious importance of heterodimerization in erbA receptor physiology, relatively little is known about the specific receptor regions that mediate this event. We (3, 24) and others (6) have shown previously that a 20-amino acid region within the ligand-binding domain of rat TRP (amino acids 286-305) must be intact for the TRP-TRAP interaction to occur. Moreover, TRsP with mutations in this 20-amino acid domain demonstrated marked functional impairment when transiently cotransfected into JEG-3 cells with a TRE- driven reporter construct. Because ligand binding, nuclear localization, and basal DNA binding were unaffected by these mutations, it was concluded that the inability to trans-activate was a direct consequence of disrupting the TRP-TRAP inter- action in vivo.

This 20-amino acid region is highly conserved throughout the erbA superfamily (Fig. 1). Therefore, we sought to inves- tigate the role of this region in mediating heterodimerization of RARa and VDR with TRAP from JEG-3 cells as well as with in vitro synthesized RXRP. In the experiments described below, we demonstrate that this 20-amino acid region in TRP, RARa, and VDR plays a critical role in receptor heterodi- merization, but that the homologous region of RXRP plays little, if any, role in this process.

MATERIALS AND METHODS

erbA cDNAs and In Vitro Mutagenesis-Wild-type and mutant rat TRsB (24,25), human RARa (26), human VDR (27), and rat RXRB (17) have been described. Oligonucleotide-directed mutagenesis of RARa, VDR, and RXRB was performed on uracil-containing DNA with the Muta-Gene M13 in vitro mutagenesis kit according to the manufacturer’s protocol (Bio-Rad). Mutations were confirmed by dideoxynucleotide sequencing.

Cell Culture and Transfections-JEG-3 cells were grown in 90% Eagle’s medium plus 10% fetal bovine serum and were transfected using standard calcium phosphate precipitation (25). All receptors were expressed from the vector pCDM (25). Transfections included

11534

erbA Superfamily Heterodimerization 11535

hVDR 2 4 4

rRXRI3 271

FIG. 1. Conservation of 20-amino acid sequence in ligand- binding domain of erbA superfamily members studied in this report. The one-letter amino acid code is used, and the amino acid numbers for each receptor are given. Residues in boldface type are identical with respect to TRB. Residues subjected to point mutation in this study are boxed. r, rat; h, human.

wild-type or mutant pCDMTR0 (3 pg), pCDMRXRP (3 pg), pCDMVDR (50 ng), or pCDMRARa (25 ng) as indicated. Expression plasmid doses were chosen because preliminary experiments indicated that they yielded near-maximal responses while still remaining on the linear portion of the dose-response curve. Vector pCDM was added where needed to achieve a total of 6 pg of pCDM-based plasmid/transfection. Ligand-responsive reporter plasmids were used at a dose of 4 pg/transfection. The reporter plasmid pTK35BA contains two copies of a modified rat growth hormone (GH) gene TRE/RARE (including the direct and inverted repeats) 5' to a basal thymidine kinase promoter directing chloramphenicol acetyltransfer- ase (CAT) expression (13). The plasmid pTK14AA contains two copies of a perfect directly repeated TRE (also derived from the rat GH gene) 5' to the thymidine kinase promoter (25). To assess vitamin D responsiveness, a VDRE consisting of two copies of the sequence 5'-GATCCACTAGGTCAAGGAGGTCATTACTGACCTCTC was inserted 5' to the same thymidine kinase promoter (pTKD3AA). To control for transfection efficiency, each transfection also included 2 pg of pTKGH, in which the basal thymidine kinase promoter directs expression of human GH. Cells in 60-mm Petri dishes were trans- fected in the presence of 10% charcoal-stripped fetal bovine serum supplemented with 100 nM dexamethasone and were then cultured for 2 days in the absence and presence of receptor-saturating doses of the appropriate ligand (100 nM T3, 1 p M all-trans-retinoic acid, or 30 nM la,25-(OH)zD3). Transfections involving all-trans-retinoic acid and la,25-(OH)2D3 were done under conditions of subdued light. Cells were harvested for CAT assay and media for human GH assay as described (25). Ligand responsiveness is defined as CAT/human GH for cells cultured with ligand divided by CAT/human GH for cells cultured without ligand. Results are the mean * S.E. for at least four transfections/condition.

Ligand Binding Assays-Following transfection of JEG-3 cells with pTKGH plus either wild-type or mutant pCDMRARa plasmids and 2 days of culture in the presence of 10% charcoal-stripped serum, the medium was replaced with 10% stripped serum + all-tran~-[~H]RA (Du Pont-New England Nuclear; 1824 GBq/mmol) k nonradioactive RA as necessary for the determination of nonspecific binding. Doses of [3H]RA ranging from 1.4 to 11.4 nM were used, with 1 p~ nonra- diolabeled RA added for determination of nonspecific binding. Incu- bations were continued for 2 h since preliminary studies indicated that binding equilibrium was achieved at that time. Media were aspirated, and the cells were washed once with ice-cold phosphate- buffered saline and once with ice-cold hypotonic TM buffer (25 mM Tris, pH 7.8, 2 mM MgC12). Cells were lysed in 0.3 ml of ice-cold hypotonic TM, 0.5% Triton X-100 and scraped into iced microcen- trifuge tubes. Nuclei were pelleted at 1000 X g at 4 "C for 10 min and washed once with ice-cold hypotonic TM buffer, and the associated radioactivity was determined. The [3H]RA binding data were nor- malized for transfection efficiency by measuring expression of co- transfected human GH.

To compare ligand affinity for wild-type and mutant VDRs, JEG- 3 cells were transfected with pTKDSAA, pCDMVDRs and pTKGH and then incubated with graded doses of h ~ , 2 5 - ( 0 H ) ~ D ~ (0 and 0.01- 300 nM in semilog doses). The ED60 for stimulation of CAT activity was taken as a measure of receptor affinity for ligand. This method was used due to the high cost of ~ ~ , ~ ~ I - [ ~ H ] ( O H ) ~ D ~ . The dose of la,25-(OH)2D3 used in all subsequent CAT induction experiments (30 nM) was chosen because it represented an EDlw for wild-type and mutant VDRs based upon these studies.

Preparation of J E G J Extract-JEG-3 cells were maintained as described above, and cell extracts were prepared as described previ- ously (3). Protein concentration was determined according to the method of Bradford (28).

Electrophoretic Mobility Shift Assay (EMSA)-~-[~S]Methionine-

labeled proteins were synthesized by in vitro translation in rabbit reticulocyte lysate (25). Products were analyzed by SDS-polyacryl- amide gel electrophoresis (PAGE) and shown to be of appropriate size (data not shown). Protein-DNA binding reactions were per- formed in 35 pl of 20 mM HEPES, pH 7.8, 50 mM KC], 1 mM dithiothreitol, 0.1% Nonidet P-40, and 20% glycerol. Incubations included 15,000-20,000 trichloroacetic acid-precipitable cpm of trans- lation products, 1.4 pg of poly(dI.dC), and 40 ng of oligonucleotide DNA (see below), either with or without 10 pg of extract from JEG- 3 cells or 15,000 cpm equivalents (by volume) of nonradiolabeled in vitro translated RXRP. Reactions were incubated at room tempera- ture for 40 min prior to electrophoresis on 0.25 X TBE (22 mM Tris base, 22 mM boric acid, 0.5 mM EDTA), 8% polyacrylamide gels (29:l acrylamide/bisacrylamide) at 4 "C. Gels were fixed, dried, and ana- lyzed by fluorography (AutofluorTM, National Diagnostics, Inc.).

Oligodeoxynucleotides used in EMSA reactions included the rat GH TRE (bp -188 to -160) (29), a synthetic palindromic TRE (30), the human osteocalcin VDRE (bp -513 to -493) (31), and irrelevant DNA from chicken actin exon 3 (bp 2096-2128).

To test whether JEG-3 cell TRAP may be an RXR, EMSA studies were also performed with the addition of RXR or control antibodies. For this purpose, mouse ascites containing RXR monoclonal antibody RX-1D12 was used at a final dilution of 150. This antibody, which is an IgG directed against the ligand-binding domain of RXR, reacts specifically with RXRa, RXRP, and RXRy.' Control ascites contain- ing a low density lipoprotein receptor IgG monoclonal antibody also was used at a dilution of 150.

Northern Blot Analysis-Total RNA was isolated from JEG-3 cells by guanidine thiocyanate/phenol/chloroform extraction (32), and poly(A)-enriched RNA was then isolated by oligo(dT)-cellulose chro- matography (33). Five pg of this RNA were electrophoresed through a 1.2% agarose-formaldehyde gel and transferred to a nylon mem- brane using standard techniques (33). A parallel lane of total RNA was electrophoresed and stained with ethidium bromide to permit the use of 28 S and 18 S ribosomal RNAs as size markers. The RNA blot was probed sequentially with random-primed 32P-labeled cDNA frag- ments encoding the amino-terminal domains of mouse RXRa, RXRP, and RXR-y (14). The cDNA for mouse RXRa included bp 96-428, that for RXRP included bp 72-363, and that for RXR-y included bp 36-344. Prehybridization was in 50% formamide, 6 X SSC (1 X SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7), 5 X Denhardt's solution (0.1% each Ficoll 400, polyvinylpyrrolidone, and bovine serum albumin), 0.2% SDS, and 100 pg/ml sheared denatured herring sperm DNA at 42 "C for 4 h. Hybridization was at 42 "C overnight and included a similar buffer except for the addition of 10% dextran sulfate and lo6 cpm/ml denatured probe. After hybridization, the final wash was in 0.1 X SSC, 0.1% SDS at 65 "C, and autoradiography was performed overnight at -70 "C with an intensifying screen.

RESULTS

TRP 20-Amino Acid Domain Mutations Impair Interaction with TRAP and RXRP-Using the avidin-biotin complex to DNA assay, TRAP from JEG-3 cells has been shown to enhance DNA binding by in uitro translated 35S-labeled TRP, and the requirement for an intact TRP 20-amino acid domain (amino acids 286-305) has been demonstrated (3,6). Although it is a very sensitive assay for protein-DNA interactions, the avidin-biotin complex to DNA assay does not allow visuali- zation of distinct complexes in a receptor-TRAP-DNA bind- ing reaction. We therefore sought to use the EMSA to identify the various species formed by coincubating these factors. Additionally, the discovery of RXR as a TRAP allowed us to compare the role of the 20-amino acid domain in TRP-JEG- 3 cell TRAP uers'sus TRP-RXR interactions. Fig. 2a demon- strates an experiment that addresses these issues. Wild-type TRP, TRP containing a deletion of the 20-amino acid region (TRpA20), and TRP harboring a point mutation that changes a highly conserved aspartate residue t o an alanine (TRPD300A) were translated in vitro in the presence of [35S] methionine. These proteins were incubated with a nonra- dioactive palindromic TRE/RARE or irrelevant DNA from

P. Chambon, personal communication.

11536 erbA Superfamily Heterodimerization a

35S-TRO: wi + A20 + D300A + Partner: - -

DNA: pal act lane: 1 2

- JEG

3 4 "

JEG RXR JEG RXR JEG RXR JEG RXR pal pal act act pal

6""- 7 8 9 10 11 12-

FIG. 2. EMSA analysis of DNA binding of wild-type and mutant TRB proteins in the presence and absence of JEG-3 extract or RXRP. a, in vitro translated wild-type (ut) ""S- labeled TRP, TRpA20, or TRPD3OOA was incubated with a nonradioactive pal- indromic TRE/RARE ( p a l ) or irrele- vant DNA from the chicken actin gene (act) (except in lunes 3 and 4 (no DNA)) f JEG-3 extract as a source of TRAP or nonradioactive in vitro translated RXRP as indicated. Radiolabeled protein-DNA complexes were separated by nondena- turing PAGE and visualized by fluorog- raphy. The radioactivity at the top of each lane represents free TRP that barely enters the gel. b, similar to a, except that incubations included i n oitro translated wild-type "S-labeled RXRB ? nonradioactive in vitro translated wild-type TRP, TRPAPO, or TRPD300A.

b [35S]RXRD: +

TREpal: - unlabeled TRD: -

Lane: 1 w

+

wt 2 0

+ + + + + + + +

wt A20 D300A 3 4 5 6 ""

the chicken actin gene (except in lanes 3 and 4 (no DNA)) using JEG-3 extract as a source of TRAP or nonradioactive in vitro translated RXRP. Using the palindromic TRE, radi- olabeled TRP is retained at the top of the gel in the absence of TRAP or RXRP (lane 1 ); the smear seen at the bottom of the lane is nonspecific as it appears when irrelevant DNA is used (lane 2 ) and in the absence of DNA altogether (lane 3 ) . The nature of this nonspecific smear is not known, but it is seen only with some batches of in vitro translated TRP (for example, it is virtually absent in Fig. 4 (lanes 1 and 10)) . The absence of a TRP monomer-TRE complex (lane 1 ) presum- ably reflects the low affinity of TRP alone for TREs as well as the small molar quantity of TRP used in this assay. The addition of JEG-3 extract in the absence of DNA (lane 4 ) causes the nonspecific smear to disappear, probably through an aggregatory mechanism. In the presence of the TRE, the addition of JEG-3 extract (lane 5 ) or RXRP (lune 6) induces the formation of a specific complex, which is not seen when irrelevant DNA is used (lanes 7 and 8) . A doublet appears in lane 5; the presence of the lower band is correlated with storage of JEG-3 extract a t -70 "C and therefore probably does not represent a unique TRAP-like factor in those ex- tracts (data not shown). Notably, specific complex formation is absent when TRPA20 is substituted for wild-type TRP (lanes 9 and 10) and reduced when TRPD300A is tested (lanes 11 and 12) .

When the [35S]methionine label is switched to in vitro translated RXRP and heterodimer-DNA interactions are ana- lyzed by EMSA, a similar story unfolds (Fig. 2b). Radiolabeled RXRP is retained at the top of the gel in the absence of DNA (lunes 1 and 2 ) or unlabeled TRP (lane 3 ) . In the presence of both the TRE/RARE and nonradiolabeled TRP as a hetero- dimerization partner, an RXRP-containing complex is noted (lane 4 ) . When nonradiolabeled TRPA20 is used in place of wild-type TRP, this complex is absent (lune 5 ) . The complex is visible when TRPD300A is used, but the magnitude of the TRP-RXRP interaction is reduced (lane 6). As expected, when the reactions in Fig. 2 (a , lane 6; and b, lane 4 ) are run side by side on the same gel, the complexes co-migrate (data not shown), indicating that these complexes contain both TRP and RXRP. These experiments demonstrate that TRAP from JEG-3 cells and RXRP behave similarly with regard to a requirement for the 20-amino acid sequence of TRP and are consistent with the notion that JEG-3 TRAP may indeed be an RXR (see below).

RARa and V D R 20-Amino Acid Domain Mutations Impair in Vitro Heterodimerization with TRAP from JEG-3 Cells and RXRP-EMSA analysis of 3sS-labeled RARa demonstrated binding to the rat GH gene TRE/RARE, but only in the presence of an auxiliary protein (TRAP) present in extracts from JEG-3 cells (Fig. 3a, lane 3 ) or i n vitro translated RXRP

erbA Superfamily Heterodimerization 11537

FIG. 3. EMSA analysis of DNA binding of wild-type and mutant RARa proteins (a) or wild-type and mutant VDR proteins (6) in the presence and absence of JEG-3 ex- tract or RXRB. a, in vitro translated wild-type (ut) %-labeled RARa, RARaA20, or RARaD256A was incu- bated with the nonradioactive TRE/ RARE (except in lane I (no DNA)) * JEG-3 extract as a source of TRAP or nonradioactive in vitro translated RXRP. Radiolabeled protein-DNA com- plexes were separated by nondenaturing PAGE and visualized by fluorography. The radioactivity at the top of each lane represents free RARa that barely enters the gel. b, similar to a, except that incu- bations included wild-type 35S-labeled VDR, VDRAZO, VDRD258A, or VDRI248S; and the specific DNA ele- ment was the human osteocalcin VDRE.

a RARawt RARaA20 RARaD256A Partner:JEG - JEG RXR - JEG RXR - JEG RXR

b

VDRwt VDRA20 VDRD258A VDR1248S Partner: - - JEG RXR - JEG RXR - JEG RXR - JEG RXR

Lane: 1 2 3 4 5 6 7 8 9 10 11 12 13 D N A : - + + + + + + + + + + + +

- - n w w o - ~ w ~ o - -

(lane 4 ) . To investigate the function of the conserved 20- amino acid domain in RARa, two mutant RARa cDNAs were created. In one mutant (RARaAZO), the entire 20-amino acid region was deleted (residues 242-261 of RARa); and in the other, the aspartic acid residue a t position 256 was changed to alanine (RARaD256A). This aspartate residue is homolo- gous to TRP Asp300. When these mutant RARs were expressed and tested in the EMSA, they did not demonstrate heterodi- merization or enhanced DNA binding in the presence of either JEG-3 cell extract (lanes 6 and 9) or RXRP (lanes 7 and 10).

Like RARa, VDR binding to DNA has been shown to be enhanced by a protein in nuclear extracts (including JEG-3 cell extracts) as well as by RXRP (17). Mutations were made in human VDR that deleted the 20-amino acid domain (resi- dues 244-263, VDRA2O) or that introduced point mutations within this region. The previously mentioned conserved as- partic acid residue was altered to alanine (VDRD258A); and another conserved residue, the isoleucine a t position 248, was changed to a serine (VDRI248S). Wild-type 3sS-labeled VDR can be induced to bind to the VDRE from the human osteo- calcin gene in the presence of either TRAP (Fig. 3b, lane 3) or RXRP (lane 4 ) . Parallel studies with 35S-labeled RXRP and unlabeled VDR demonstrated that the complex contains both proteins (data not shown). Ablation of the VDR 20- amino acid domain is sufficient to prevent RXRP and TRAP from interacting with VDR (lanes 6 and 7). Mutation of the conserved aspartic acid residue in VDR attenuates the inter- action with TRAP and RXRP, but does not prevent it (lanes 9 and IO); the 1248s mutation, however, disrupts this inter- action fully (lanes 12 and 13).

JEG-3 Cell TRAP May Be RXRa-The above data indicate that mutations in TRP, RARa, and VDR that disrupt heter- odimerization with JEG-3 cell TRAP also disrupt heterodi- merization with RXRP, raising the question of whether JEG- 3 cell TRAP really is an RXR. To address this, we first probed a Northern blot of JEG-3 cell poly(A)-enriched RNA with probes specific for RXRa, RXRP, and RXRr. The RXRa probe hybridized primarily to a 5-kilobase RNA (data not shown), consistent with the reported size of rat RXRa mRNA (22, 34). However, no signal was seen with the RXRB and RXRr probes (data not shown). Thus, a t the RNA level,

JEG-3 cells express primarily RXRa. To assess whether JEG-3 cell TRAP may be RXRa, an

EMSA was performed using '%-labeled TRP and TRAP with or without a monoclonal antibody directed against the ligand- binding domain of RXR. This monoclonal antibody binds specifically to all three RXRs.' Using a palindromic TRE, the typical TRP. TRAP. TRE complex is visualized (Fig. 4, lane 4 ) . Addition of the RXR antibody to the reaction mixture prevents formation of this complex (lane 5), presumably because the antibody binds to TRAP and sterically precludes TRP binding. The control monoclonal antibody does not affect the TRP. TRAP. TRE complex (lane 8). Similar results are seen when nonradiolabeled in vitro synthesized RXRP substitutes for TRAP. Thus, the TRP. RXRP.TRE complex is visualized in lane 6; and formation of this complex is blocked by the RXR antibody (lane 7), but not by the control antibody (lane 9).

To demonstrate that the antibody is truly reacting with RXR and not TRP, an EMSA was performed using a directly repeated TRE with a 4-bp spacer (35), on which we can observe %labeled TRP binding in the absence of RXR/ TRAP. The TRP.TRE complex is seen in Fig. 4 (lane 11 ); this complex is unaffected by addition of the RXR antibody (lane 12). Therefore, as expected, the inhibition of complex formation seen in lanes 5 and 7 is likely to be due to antibody binding to RXR. Overall, the Northern blot and EMSA data are consistent with JEG-3 cell TRAP being an RXRa.

20-Amino Acid Domain Mutations Impair Functional Activ- ity of RARa and VDR-Mutations that disrupt heterodimer- ization in vitro provide a mechanism for studying the impor- tance of heterodimerization for receptor function in vivo. Toward this end, we transfected wild-type and mutant RARa and VDR cDNAs into JEG-3 cells and assessed their ability to trans-activate appropriate reporter constructs in the pres- ence of ligand. Using the reporter plasmid pTK35BA, cotrans- fection with wild-type RARa led to a 45-fold induction of CAT in the presence of RA (Fig. 5a). In contrast, transfection of RARaD256A in place of wild-type RARa led to only a 15- fold induction in response to RA. In the absence of either exogenous RARa or the RARE, CAT expression was essen- tially unresponsive to RA.

11538 erbA Superfamily Heterodimerization

['%]TRO: + + + + + + + + + Partner: - JEG - JEG JEG RXR RXR JEG RXR - - - + + +

DNA: - - Pal Pal Pal Pal Pal Pal Pat - DR4 1334 FIG. 4. Antibody-mediated inhi- Antibody: - - - - R X - R X C C - - R X bition of TRP-TRAP heterodimeri- Lane: 1 2 3 4 5 6 7 8 9 10 11 12 zation. In vitro translated :"S-labeled TRB was incubated with a TRE (except in lanes 1 ,2 , and 10 (no DNA)) f JEG- 3 extract as a source of TRAP or non- radioactive in vitro translated RXRP as indicated. Incubations also included a monoclonal antibody specific for RXRs ( R X ) or a control antibody ( C ) where indicated. Samples were electrophoresed as described for Figs. 2 and 3. Lanes 3-9 utilized a palindromic TRE (pal), and lanes 11 and 12 utilized a direct repeat TRE with a 4-bp spacer. Lanes 10-12 were run on a separate gel.

""" "- no="

c 40-

2 8 30-

z 20- 9 0 L L

10-

0- I I -

RAR: none wild type D256A wild type RARE: TK35BA TK35BA TK35BA none

b 10

c

3 6 a 0 4

l-

9 0 LL

2

C i T

VDR: wild type D258A none wild type VDRE: TKD3AA TKD3AA TKD3AA none

FIG. 5. Functional activity of wild-type and mutant RARw (a) or VDRs (b ) . a, JEG-3 cells were transiently transfected with an expression vector containing wild-type RARa, RARaD256A, or no insert, along with an RARE/CAT reporter plasmid (pTK35BA) or the reporter plasmid without an RARE. Transfected cells were incubated for 2 days in the presence and absence of 1 PM all-trans- retinoic acid, harvested, and assayed for CAT expression. Transfec- tion efficiency was standardized using an internal control plasmid expressing human GH. b, similar to a, except that transfections included wild-type VDR, VDRD258A, or empty vector, along with a VDRE/CAT reporter plasmid (pTKD3AA) or the reporter plasmid without the VDRE. Transfected cells were incubated for 2 days in the presence or absence of 30 nM la,25-(OH)*D3 prior to harvest.

Since the 20-amino acid domain lies within the ligand- binding domain, it was important to show that the deficiency in trans-activation was not due to diminished ligand binding. For RARaD256A, this was tested by measuring [3H]RA bind- ing in the nuclei of transfected cells. Scatchard analysis indicated that RARaD256A and wild-type RARa had identi- cal dissociation constants for ligand (4 nM in the presence of 10% serum). In addition, the nuclear maximal binding capac-

ity for ["IRA was comparable for both receptors: 10 fmol of ['H]RA/pg of human GH for wild-type RARa and 12 fmol of [3H]RA/pg of human GH for RARaD256A (versus 3.7 fmol of [3H]RA/pg of human GH for cells transfected with empty vector). Therefore, we conclude that the deficiency in truns- activation associated with RARaD256A cannot be explained by diminished ligand binding or nuclear expression of this transfected receptor.

Transfection studies were also performed using wild-type and mutant VDRs. As seen in Fig. 5b, la,25-(OH)2Ds was able to induce CAT expression driven by a VDRE 9-fold in the presence of wild-type VDR. The mutation at AspzsR only reduced this induction slightly, to just over 6-fold. This mod- est reduction is in keeping with the EMSA data, which indicate that this residue is less critical for heterodimerization of VDR than is the corresponding residue in RARa. However, even though the dose of vitamin D used in these studies (30 nM) represented an EDI, for CAT induction by both the wild-type and mutant VDRs (data not shown), the EDsO values for these receptors were slightly different (0.3 nM la,25- (OH)*D3 for wild-type VDR and 1.0 nM for VDRD258A). Despite the use of a receptor-saturating dose of 1a,25- (OH)zD3, it would be difficult to exclude subtle defects in the ligand binding or nuclear expression of VDRD258A as expla- nations for the modest decrement in vitamin D responsiveness observed with this mutant receptor.

In contrast to VDRD258A, preliminary experiments indi- cated that VDRI248S does not bind la,25-['H](OH)2D3 (data not shown). Therefore, even though the 1248s mutation dis- rupted heterodimerization in vitro, it was not possible to assess the effects of that disruption on transcriptional acti- vation.

Taken together, the above data indicate that the 20-amino acid domain of TRP, RARa, and VDR provides a critical interface for heterodimerization with TRAP from JEG-3 cells as well as with in vitro translated RXRP. Furthermore, dis- ruption of this domain is sufficient to impair the transcrip- tional response to ligand by mutant receptors, thus suggesting a major role for heterodimerization with RXR or perhaps other TRAPS in trans-activation by these receptors.

Effects of Mutations in 20-Amino Acid Domain of RXRP on Its Interaction with Other erbA Superfamily Members-The discovery that RXR possesses TRAP activity, coupled with its identity as an erbA superfamily member, raises the possi- bility that heterodimerization involves the same domains of RXR as TRs, RARs, and VDR. To test this hypothesis, we

erbA Superfamily Heterodimerization 11539

constructed RXRP cDNAs that encode proteins with either a deletion of the 20-amino acid region (residues 271-290 of rat RXRP, RXRPA2O) or point mutations a t 3 highly conserved residues (A272D, I275S, and D285A). Homologous point mu- tations in TRP (3, 24), RARa, and/or VDR disrupt heterodi- merization.

Analysis by EMSA reveals that none of these point muta- tions in RXRP disrupts heterodimerization with TRP (Fig. 6, lane 4 versus lanes 6,8, and 10). In fact, deletion of the entire 20-amino acid domain of RXRP diminishes, but does not abolish, heterodimerization with TRP (lane 12). These studies were performed on a palindromic response element, but iden- tical results were achieved with a direct repeat TRE (data not shown). Similar studies were performed with RARa as the heterodimerization partner on the palindromic TRE/RARE. Again, the RXRP point mutations failed to affect heterodi- merization, although the deletion mutation totally abolished interaction with RARa (data not shown).

These results are supported by data from gene transfer experiments in which JEG-3 cells were transiently cotrans- fected with TRP and wild-type or mutant RXRP cDNAs. Previously, it has been shown that wild-type RXRP enhances the ability of TI to induce expression of reporter genes driven by direct repeat TREs (14, 15, 17-19, 23, 36). In concert with this, we observed a 15 k %fold TB induction of CAT following transfection with TRP and the reporter plasmid pTK14AA, but a 49 k 5-fold T3 induction if the transfection also included wild-type RXRP ( n = 4). RXRPD285A was as effective as wild-type RXRp at enhancing T3 induction of CAT (56 +. 5- fold induction), whereas RXRpA20 yielded no increase in TB induction over that observed in the absence of cotransfected RXR (15 k 1-fold induction).

DISCUSSION

Many members of the erbA superfamily of ligand-inducible transcription factors are believed to interact with specific DNA sequences, or response elements, as homodimers. This notion was suggested by the dyad symmetry of steroid re- sponse elements and has been supported by a wide variety of data. Homodimers of estrogen receptors, for example, have been shown to exist in solution (37), and cooperative binding of two glucocorticoid receptor DNA-binding domains to a glucocorticoid response element has been demonstrated (38). Domains responsible for dimerization have been localized to a t least two distinct regions of the glucocorticoid and estrogen receptors. A weak contact site exists within the DNA-binding domain that probably helps to determine spacing require- ments for the receptor complex on the DNA (39). A much more potent dimerization determinant is located in the ligand- binding domain and has been mapped in the estrogen receptor

to a stretch of amino acids in the distal COOH terminus (40). The subgroup of erbA receptors that includes the TRs,

RARs, and VDR, however, has not been demonstrated to exist as dimers in solution; and the only evidence for homodimeri- zation on DNA comes from EMSA analysis (41, 42). Inter- pretation of these data is complicated as it is difficult to distinguish a true dimer from two receptors that bind to the same piece of DNA but do not physically contact one another. In contrast to this, a physical interaction between this group of receptors and an auxiliary protein (TRAP) from nuclear extract has been demonstrated (3, 42). The identification of the RXR family and the subsequent realization that these erbA superfamily proteins could substitute for TRAP from nuclear extract provided insight into a mechanism of trans- activation by nuclear receptors that involves heterodimeriza- tion.

Despite the growing realization of the importance of this interaction, very little has been done to map specific regions of these receptors that are involved in the heterodimerization event. Several groups have pointed out that large COOH- terminal deletions of TRs and RARs render those receptors unable to interact with TRAP or RXR (5, 14, 17), and Glass et al. (8) have shown that the ligand-binding domain of RARa is sufficient to be cross-linked to auxiliary proteins from HeLa and HL-60 cells. Forman and Samuels (43) have proposed that TRs, RARs, and VDR belong to a subclass of erbA proteins that share nine “heptad repeats” in the ligand-bind- ing domain that are reminiscent of leucine zippers and helix- loop-helix motifs, known dimerization domains for other classes of transcription factors. Important differences exist, however; classic leucine zippers have four continuous heptad repeats organized into a single a-helix, whereas these erbA proteins have nine heptad repeats organized into five discon- tinuous groups of unknown secondary structure. Moreover, leucine zippers contain only leucine residues at the critical I-, 5-, and 8-positions that present themselves as a hydrophobic helical face (44). The corresponding situation in erbA recep- tors is less clear-cut; positions 1 and 8 may be held by hydrophobic, non-leucine residues (e.g. Ile, Val, Met, and Phe), whereas position 5 may be occupied by either hydro- phobic residues or charged amino acids with hydrophobic side chains (Arg and Gln). Also, leucine zipper-mediated dimeri- zation is absolutely required for DNA binding by proteins with these motifs (45); this is not the case for nuclear recep- tors. Thus, the heptad repeats of erbA receptors appear to be related to, but distinct from, classical leucine zippers. In any case, studies using COOH-terminally truncated erbA family proteins support a role for sequences at the distal COOH terminus of several erbA receptors, including the ninth of these heptad repeats, in the interaction with TRAP (14, 46).

[35S]RXRO: wild type A272D 12758 D285A A20 Cold TRB: - + + + + + +

DNA: - + + + + + Lane: 1 2 3 4 5 6 7 8 9 10 11 12

+ + + + +

FIG. 6. EMSA analysis of DNA binding of wild-type and mutant RXRB proteins in presence and absence of nonradiolabeled TRB. In vitro translated wild-type ”S-labeled RXRP, RXRPA272D. RXRPI275S, RXRPD285A, or RXRPASO was incubated with a nonradioactive palindromic TRE (except in lanes I and 2 (no DNA)) ? nonradioactive in vitro translated TRP. Radiolabeled protein-DNA complexes were separated by nondenaturing PAGE and visualized by fluorography.

11540 erbA Superfamily Heterodimerization

In addition, Spanjaard et al. (47) have focused on a putative amphipathic helix in TRal (amino acids 288-331) that over- laps with the second and third heptad repeats. Mutations in this region decrease the ability of TRAP to enhance DNA binding by these proteins. The importance of this region in allowing heterodimerization-mediated trans-activation in vivo cannot be tested directly because ligand binding is disrupted in addition to protein-protein interactions. In addition, the disruption of ligand binding makes it somewhat more difficult to exclude a role for distant conformational changes account- ing for the mutant phenotype.

While deletional analysis suggests a role for distal COOH- terminal structures in heterodimerization, evidence also exists to support a role for sequences more NHz-terminal in the ligand-binding domain. O'Donnell and Koenig (24) investi- gated the function of a 20-amino acid segment from the ligand-binding domain of rat TRP (amino acids 286-305) (Fig. 1) that is highly conserved among all members of the receptor superfamily despite relatively low homology throughout the rest of the ligand-binding domain. This region is located NHz- terminal to the first heptad repeat. Mutations of this segment were found to impair the ability of TRP to trans-activate in transfected JEG-3 cells. No defects in ligand binding, nuclear translocation, or basal DNA binding were identified to ac- count for the inability to trans-activate. However, these 20- amino acid domain mutations did impair the TRP-TRAP interaction (3, 6). The studies presented herein demonstrate that this region also is involved in TRP-RXRP heterodimeri- zation. Indeed, a combination of Northern analysis and EMSA with an RXR antibody supports the notion that JEG- 3 cell TRAP may be an RXRa. Further studies in this report demonstrate that the homologous 20-amino acid regions of RARa and VDR also are involved in heterodimerization, both with JEG-3 cell TRAP and RXRP. In any study involving mutant proteins, there is the possibility that the effects noted are due to secondary conformational changes at sites distant from the mutation itself. However, the fact that ligand binding is wild type for TRPD300A (24) and RARaD256A makes a global disruption of conformation unlikely. Furthermore, since receptors with mutations in this domain still bind ligand, one can use transfection to test the consequences of these mutations on reporter gene activation. These transfection studies indicate that not only do the 20-amino acid domains of TRP, RARa, and VDR play critical roles in heterodimeri- zation with TRAP (or RXR), but also that these interactions are required for full trans-activation by these receptors. In contrast, mutations in other regions of these receptors that disrupt heterodimerization also disrupt ligand binding (see above); and therefore, the consequences of those mutations cannot be tested by transfection analysis.

Since RXR also is an erbA superfamily member, one might expect that RXR would utilize the same 20-amino acid and heptad domains that TR, RAR, and VDR use in heterodimer- ization. Indeed, deletion of either the ninth heptad repeat (14) or the 20-amino acid domain of RXR is sufficient to impair such protein-protein interactions. However, these results could simply reflect gross conformational changes brought about by the deletions. Indeed, at least for the 20-amino acid domain, the lack of effect of three point mutations within this region would support this interpretation. Even if the 20-amino acid domain of RXR is involved in heterodimerization, it must play a secondary, nonessential role. Thus, the hetero- dimerization of RXR with other erbA superfamily members must be an asymmetric process, with the two heterodimeri- zation partners utilizing distinct regions of their ligand-bind- ing domains as contact points.

Even though the 20-amino acid domains of TRP, RARa, and VDR all play important roles in heterodimerization, it appears that the specific contact points may differ with each receptor. Thus, for example, an identical aspartate-to-alanine mutation has major functional consequences when found in TRP or RARa, but modest consequences when found in VDR. In addition, it should be noted that there is a less than perfect correlation in the functional consequences of receptor muta- tions when studied by EMSA versus transfection. On the one hand, these correlations are excellent when studying the effects of the aspartate-to-alanine mutation in TRP, RARa, or RXRP. However, the same mutation in VDR has a sub- stantially more disruptive effect when studied by EMSA than by transfection. The reason for this is not known, but it is possible that heterodimers that are unstable under the con- ditions of EMSA are sufficiently stable in a transfected cell to induce gene expression.

Many other classes of transcription factors, such as the AP- 1 family (e.g. c-Fos and c-Jun) and rel-related proteins (NF- KB, p50, and p65), have been shown to function as heterodi- mers (48,49). A large body of evidence has recently accumu- lated that indicates that some erbA superfamily members operate in this fashion as well. It is probable that different combinations of these proteins in various complexes can exist within a single cell. This flexibility is likely to allow for exquisite sensitivity in regulating gene expression by these proteins and their ligands. By extending the types of analyses described above, we hope to be able to describe the physical heterodimer in detail at the submolecular level and to delin- eate possible mechanisms of combinatorial regulation by li- gand or other cell-specific factors.

Acknowledgments-We thank Milan Uskokovic for the 1~1,251- (OH),D,, Pierre Chambon for the RXR antibody and mouse RXR cDNAs, Chris Glass and Michael G. Rosenfeld for the rat RXRp cDNA, Ron Evans for the human RARa cDNA, J. Wesley Pike for the human VDR cDNA, and Gregory A. Brent for pTK35BA.

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