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Endocrinology Vol. 139, No. 10 4197-4204
Copyright © 1998 by The Endocrine Society

ARTICLES

Lack of Coactivator Interaction Can Be a Mechanism for Dominant Negative Activity by Mutant Thyroid Hormone Receptors1

Ying Liu, Akira Takeshita, Silvia Misiti, William W. Chin and Paul M. Yen

Division of Genetics (A.T., S.M., W.W.C.), Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115; Molecular and Cellular Endocrinology Branch (Y.L., P.M.Y.), NIDDK/National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Dr. Paul M. Yen, Molecular and Cellular Endocrinology Branch, NIDDK/National Instiutes of Health, Building 10, Room 8D04, 9000 Rockville Pike, Bethesda, Maryland 20892. E-mail: PaulY{at}Bdg10.NIDDK.NIH.Gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
We studied the interactions of two natural thyroid hormone receptor (TR) mutants from patients with resistance to thyroid hormone (RTH) and an artificial TR mutant with a nuclear receptor corepressor, N-CoR, and a steroid receptor coactivator, SRC-1. In electrophoretic mobility shift assays, wild-type TRß-1 interacted with N-CoR in the absence of ligand, whereas T3 caused dissociation of the TRß-1/N-CoR complex and formation of TRß-1/SRC-1 complex. In contrast, a natural mutant (G345R) with poor T3-binding affinity formed TRß-1/N-CoR complex, both in the absence and presence of T3, but could not form TRß-1/SRC-1 complex. Another TR mutant, which bound T3 with normal affinity and containing a mutation in the AF-2 region (E457D), had normal interactions with N-CoR but could not bind SRC-1. Both these mutants had strong dominant negative activity on wild-type TR transactivation. Studies with a TR mutant that had slightly decreased T3-binding affinity (R320H) showed a T3-dependent decrease in binding to N-CoR and increase in binding to SRC-1 that reflected its decreased ligand binding affinity. Additionally, when N-CoR and SRC-1 were added to these receptors at various T3 concentrations in electrophoretic mobility shift assays, TR/N-CoR and TR/SRC-1 complexes, but not intermediate complexes were observed, suggesting that N-CoR release is necessary before SRC-1 binding to TR. Our data provide new insight on the molecular mechanisms of dominant negative activity in RTH and suggest that the inability of mutant TRs to interact with coactivators such as SRC-1, which results from reduced T3-binding affinity, is a determinant of dominant negative activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
RESISTANCE to thyroid hormone (RTH) is a syndrome of hyposensitivity to T3 that usually displays a pattern of autosomal dominant inheritance (1). Recent studies of patients from families with RTH have identified nucleotide substitutions in one of their TRß alleles resulting in point mutations in the ligand binding domain and impaired T3-binding (1, 2, 3). However, because there are two normal TR{alpha} alleles and one normal TRß allele in patients with RTH, little is known about how the mutant TR exerts its dominant negative activity on wild-type (WT) TRs in T3-responsive target tissues.

Studies of natural TR mutants suggest that the dominant negative activity likely involves competition for binding to TREs by mutant homodimers that cannot bind T3 (as WT TRß homodimers normally dissociate from DNA in the presence of T3) and/or the formation of inactive mutant TR/retinoid X receptor (RXR) heterodimers (4, 5). Previous studies also have shown that many of these mutants have impaired T3-dependent transactivation, presumably due to reduced T3-binding (1, 2, 3). Additionally, similar to WT TR, these mutant receptors also exhibited repression of basal transcription in the absence of T3 (6).

Recently, several groups have identified putative corepressors (N-CoR, SMRT) that may mediate basal repression by unliganded TRs (7, 8, 9, 10) as well as putative coactivators (e.g. SRC-1; TIF2; RIP 140; p/CIP; RAC3/ACTR/TRAM-1/AIB1) that may mediate ligand-dependent transactivation by TRs and other nuclear hormone receptors (11, 12, 13, 14, 15, 16, 17, 18, 19). In this paper, we have examined the interaction of two natural mutant TRs and an artificial mutant TR containing an amino acid substitution in the AF-2 transactivation domain (20, 21) with a putative corepressor, N-CoR and a coactivator, SRC-1. Our findings demonstrate that these receptors have impaired interaction with SRC-1 that correlates with their defective transcriptional and dominant negative activities in cotransfection assays. Taken together, these results suggest that inability to interact with coactivators such as SRC-1 can be a determinant of dominant negative activity by mutant TRs in RTH.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Preparation of receptors and cofactors
Complementary DNA (cDNA) clones of hTRß-1, R320H, and G345R cloned in pcDNA were used in these assays as previously described (6, 22). A cDNA clone encoding amino acids 1539–2453 of human N-CoR (N-CoRI) in pKCR2 also was used (kindly provided by Dr. A. Hollenberg, Beth Israel Deaconess Medical Center, Boston, MA) (23). The AF-2 mutant, E457D, was created by PCR using a primer containing a mutation of the codon E457D and HindIII restriction site, and a primer containing a SmaI restriction site and the rat TRß cDNA containing the first translational start site methionine. The PCR fragment was isolated, purified, and then subcloned into the pcDNA expression vector. The hinge region mutant was created by site-directed mutagenesis kit (Promaga) with the codons A223G, H224G, and T227A (8). The fragment was purified and subcloned into the TRß-1 vector in pcDNA. Hinge/457 was generated by subcloning a BstX-1 fragment from the Hinge vector into the corresponding sites of the E457D vector. Proteins were generated by in vitro translation (Promega, Madison, WI) and [35S] methionine-labeled receptor protein was quantitated by SDS-PAGE analysis, which showed similar expression of labeled proteins of expected molecular weights.

A cDNA fragment of human SRC-1 encoding amino acids 595 to 780 (14) was generated by PCR, subcloned into a glutathione-S-transferase-fusion vector, and expressed in Escherichia coli. The protein was isolated and purified as previously described (24).

DNA binding assay/electrophoretic mobility shift assay (EMSA)
A deoxyribonuceotide containing the chicken lysozyme TRE, F2, was end-labeled with [32P] {gamma}-ATP by T4 polynucleotide kinase (25). In vitro translated receptor and 10,000 cpm oligonucleotide probe were mixed and incubated together before being subjected to electrophoresis and autoradiography as previously described (25).

Cotransfection studies
cDNA clones of TRß-1, R320H, G345R, and E457D described above were used in the cotransfection experiments. A previously descrbed reporter plasmid containing the F2 TRE and the luciferase cDNA in PT109 was used (26, 27).

CV-1 cells were grown in DMEM/10% FCS. The serum was stripped of T3 by incubating with charcoal for 12 h at 4 C, and constant mixing with 5% (wt/vol) AG1-X8 resin (Bio-Rad, Richmond, CA) twice for 12 h at 4 C before ultrafiltration. Unless otherwise indicated, the cells were transfected with expression (0.1 µg) and reporter (1.7 µg) plasmids as well as a RSV-ß-galactosidase control plasmid (1 µg) (28) in 3.5-cm plates using the calcium-phosphate precipitation method (29). Cells were grown for 48 h in the absence or presence of 10-6 M T3 (Sigma Chemical Co., St. Louis, MO), and harvested. Cell extracts were analyzed for both luciferase (30) and ß-galactosidase (28) activity to correct for transfection efficiency. The corrected luciferase activities of untreated samples were normalized to the luciferase activities of samples containing vector alone in the absence of ligand (1-fold basal).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
We first used EMSA to examine human TRß-1 interactions with the putative corepressor, N-CoR and the coactivator, SRC-1 on a labeled oligonucleotide containing the chicken lysozyme TRE, F2 (Fig. 1AGo). In the absence of RXR and ligand, TRß-1 bound to F2 oligonucleotide primarily as a homodimer (Fig. 1AGo, lane 2). Addition of T3 decreased TRß-1 homodimer binding to DNA (Fig. 1AGo, lane 3). In the absence of T3, TRß-1 interacted with N-CoR resulting in an additional more slowly migrating protein/DNA complex (Fig. 1AGo, lane 4). In the presence of T3, this complex also exhibited decreased binding to DNA (Fig. 1AGo, lane 5). An opposite pattern was observed for TRß-1 interaction with SRC-1 as ligand promoted formation of TRß-1/SRC-1/DNA complex (Fig. 1AGo, lanes 6 and 7). TRß-1/RXR heterodimer interacted with N-CoR in a ligand-independent and SRC-1 in a ligand-dependent manner (Fig. 1AGo, lanes 10–13). We were unable to detect TRß-1 monomer/SRC-1 complexes when we used a probe, F2M, containing only a single half-site (25) (data not shown).



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  		Figure 1. TRß-1 interaction with N-CoR and SRC-1 on the inverted palindrome F2 TRE. In vitro translated TRß-1 (1 µl), RXRß (2 µl) and N-CoR (2 µl) and/or GST-SRC-1 (20 ng) were incubated with [32P]-labeled oligonucleotide in the presence or absence of 10-7 M T3, and then analyzed by EMSA as described in Materials and Methods. Reticulocyte lysate was added to some samples so that the total volume of reticulocyte lysate was the same for each sample. A, TRß homodimer and heterodimer interaction with N-CoR and SRC-1 in the absence or presence of T3. Note there are two TRß/RXR heterodimer bands due to two in vitro translated RXR products. B, Antibody supershift of TRß-1/RXRß/SRC-1 complex on the inverted palindrome F2 TRE. Preimmune, anti-TRß-1 or anti-RXR antibodies (1 µl) were added for 2 h at 4 C after samples were incubated with probe and 10-7 M T3, and then analyzed by EMSA. SS, Supershifted complex; pre, preimmune serum; rl, reticulocyte lysate.

 
Because T3-bound TR/RXR heterodimer is presumed to be the transcriptionally active TR-containing complex for many target genes, we wanted to confirm that heterodimer interacted with SRC-1. As seen in Fig. 1BGo, anti-TRß-1 and anti-RXRß antibodies (31, 32) were both able to supershift the putative TR/RXR/SRC-1/DNA complex demonstrating that TR/RXR heterodimer can interact with SRC-1 in EMSA. Of note, Zamir et al. (33) recently showed that TR/RXR heterodimer also can interact with N-CoR and SMRT. Additionally, our observation that TRß-1 homodimer can form a complex with SRC-1 in the presence of T3 (Fig. 1AGo), suggests that SRC-1 may stabilize liganded TR binding to DNA. Previously, it was observed that T3 abrogated TR homodimer binding to TREs in vitro, suggesting that TR homodimers might participate in mediating basal repression (25). While this still may be the case, these data raise the possibility that TR homodimers also may be able to mediate T3-dependent transcription. Currently, the nature of TR complexes with cells is not known and may depend on the relative stoichiometry and compartmentalization of TRs and RXRs in the nucleus of particular cell types. These data also demonstrate that ligand-bound TR on DNA is sufficient to interact with SRC-1, and heterodimerization with RXR is not necessary for this interaction. In this connection, liganded TR previously was shown by GST pull-down studies to interact with SRC-1 in solution (16, 17).

We next examined N-CoR and SRC-1 interactions with two natural mutants from patients with RTH (G345R and R320H) and a TRß-1 mutant involving the AF-2 domain (E457D) (Fig. 2Go). G345R has virtually undetectable ligand binding affinity (Ka < 0.01 WT Ka) and is unable to transactivate in the presence of T3 (6, 17). R320H has reduced ligand binding affinity (Ka = 0.42 WT Ka) and mediates weak transactivation (6, 22). E457D has normal ligand binding affinity (Ka = 1.23 WT Ka) but is unable to transactivate, and has been considered a pure transactivation-deficient mutant (21, 34). As seen in Fig. 2Go, homodimers of these mutants bound to F2 in the absence of T3. As reported previously, the G345R homodimer was unable to dissociate from DNA in the presence of T3 (5), but R320H homodimer dissociated in the presence of high T3 concentration (Fig. 2AGo, lanes 1–3, Fig. 2BGo, lanes 1–3). E457D homodimer displayed a similar dissociation pattern as WT TRß-1 (Fig. 2CGo). G345R formed a complex with N-CoR and remained constitutively bound to N-CoR even in the presence of 10-6 M T3 (Fig. 2AGo, lanes 4–6). R320H complexed with N-CoR in the absence of T3 and dissociated from DNA at 10-7 M T3 (Fig. 2BGo, lanes 4–6). Interestingly, E457D interactions with N-CoR were similar to WT TRß-1 (Fig. 1Go and Fig. 2CGo, lanes 4–6).



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  		Figure 2. Mutant TRß-1 homodimer interaction with N-CoR or SRC-1 on F2 TRE. In vitro translated TRß-1 (1 µl) mutants (G345R, R320H and E457D) and N-CoR (2 µl) and GST-SRC-1 (20 ng) were incubated with [32P]-labeled F2 and then analyzed by EMSA as in Fig. 1Go. Reticulocyte lysate was added to some samples so that the total volume of reticulocyte lysate was the same for each sample. Same amounts of in vitro translated TRß-1 and mutants, as quantitated by SDS-PAGE analyses, were added. A, G345R; B, R320H; C, E457D.

 
We then examined these receptor interactions with SRC-1. G345R and E457D were unable to interact with SRC-1 even at 10-6 M T3 (Fig. 2AGo, lanes 7–9; Fig. 2CGo, lanes 8–10). On the other hand, R320H complexed with SRC-1 in a dose-dependent manner (10-6 M > 10-7 M T3) (Fig. 2BGo, lanes 8 and 9). Similar results also were observed when we examined TR/RXR heterodimer interactions with N-CoR and SRC-1 (Fig. 3Go, A–D). As observed for N-CoR interactions with these two receptors, the differences between R320H and G345R likely represent their differences in ligand binding affinity. In contrast, because E457D had normal ligand binding affinity, its selective inability to interact with SRC-1 (but not N-CoR) likely represented changes in protein/protein interactions with coactivator(s). In the foregoing EMSA experiments, we used truncated versions of N-CoR and SRC-1. Similar, but slightly weaker, interactions were observed between full-length versions of these cofactors (8, 17), and WT or mutant TRs (data not shown).



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  		Figure 3. Mutant TRß-1 heterodimer interaction with N-CoR or SRC-1 on F2 TRE. In vitro translated TRß-1 mutants (1 µl) (G345R, R320H and E457D), RXRß (2 µl) and N-CoR (2 µl) and GST-SRC-1 (20 ng) were incubated with [32P]-labeled F2 and then analyzed by EMSA as in Fig. 1Go. Reticulocyte lysate was added to some samples so that the total volume of reticulocyte lysate was the same for each sample. Similar amounts of TRß-1 and mutants, as quantitated by SDS-PAGE analyses, were added. 10-7 M or 10-6 M T3 were added to some samples as indicated (Log T3 = log T3 concentration). A, TRß-1 WT; B,G345R; C, R320H; D, E457D.

 
We next examined whether corepressor release was necessary for coactivator binding to WT TR and these mutants by adding both N-CoR and SRC-1 with TR, and examining the formation of TR/N-CoR and TR/SRC-1 complexes on the F2 TRE (Fig. 4Go). Increasing concentrations of T3 decreased the formation of TRß/N-CoR complex while promoting the formation of TRß/SRC-1 complex (Fig. 4AGo). At 10-8 M T3, mostly TRß/SRC-1 complexes were observed. As expected from the findings in Figs. 2Go and 3Go, G345R remained complexed to N-CoR from 10-10 M to 10-7 M T3 and was unable to form a G345R/SRC-1 complex (Fig. 4BGo). E457D/N-CoR decreased in a T3-dependent manner but was unable to form a E457D/SRC-1 complex (Fig. 4CGo). Similar to WT TRß, but at 10-7 M T3, there were approximately equal amounts of R320H/N-CoR and R320H/SRC-1 complexes reflecting the decreased ligand-binding affinity of this mutant TR (Fig. 4DGo). Interestingly, no intermediate complexes were observed, suggesting that there is a dynamic equilibrium between TRß/N-CoR and TRß/SRC-1 formation depending on T3 concentration.



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  		Figure 4. WT and mutant TRß-1 interaction with N-CoR and SRC-1 on F2 TRE. In vitro translated WT TRß-1 and mutants (G345R, R320H and E457D) (1 µl) and N-CoR (2 µl) and GST-SRC-1 (20 ng) were incubated with [32P]-labeled F2 and then analyzed by EMSA as in Fig. 1Go. Reticulocyte lysate was added to some samples so that the total volume of reticulocyte lysate was the same for each sample. Same amounts of in vitro translated TRß-1 and mutants, as quantitated by SDS-PAGE analyses, were added. (Log T3 = log T3 concentration) A, TRß-1 WT; B, G345R; C, R320H; D, E457D.

 
We next examined the transcriptional activity and dominant negative activity of these TR mutants in CV-1 cells. Northern blot analyses demonstrated that both N-CoR and SRC-1 are expressed endogenously in these cells, and thus likely function as coregulators in CV-1 cells (data not shown). TRß-1 repressed basal transcription in the absence of T3 and stimulated transcription in the presence of T3 when cells were cotransfected with TRß-1 expression vector and a F2-containing reporter vector (Fig. 5AGo). G345R had constitutive basal repression in the absence or presence of T3. These findings are consistent with the observation that G345R remained bound to N-CoR even at 10-6 M and was unable to interact with SRC-1, in EMSA (Fig. 2Go). Recently, similar to these findings, Yoh et al. showed that G345R constitutively bound to another corepressor, SMRT, even in the presence of T3 (35). E457D had normal basal repression and derepression, and no ligand-dependent transcriptional activity. These findings are consistent with formation of E457D/N-CoR complex in the absence of T3 and its dissociation from DNA in the presence of T3, as well as the inability of E457D to interact with SRC-1 on DNA. In Fig. 5BGo, R320H derepressed in the presence of T3 and had weak transactivation in the presence of 10-7 M T3. Addition of 10-6 M T3, increased transactivation. These findings are consistent with its less severe impairment of ligand-binding than G345R.



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  		Figure 5. Transcriptional and dominant negative activities of TRß-1 mutants on F2. TRß-1 WT, G345R and E457D expression vector (0.1 µg), F2 reporter plasmid (1.7 µg), and ß-galactosidase control vector (1.0 µg) were cotransfected in CV-1 cells in the absence or presence of 10-7 M or 10-6 M T3 for 48 h. A 3:1 or 5:1 ratio of mutant TRß (0.3 µg or 0.5 µg) and TRß-1WT (0.1 µg) expression vectors was added to some samples. pcDNA vector alone was added to some samples to keep total DNA constant. In these experiments, treated cells were harvested and luciferase measured as described in Materials and Methods. Luciferase activity was normalized to ß galactosidase activity and then calculated as fold basal luciferase activity with 1-fold basal activity defined as the luciferase activity with control pcDNA vector alone in the absence of ligand. Each point represents the mean of four experiments with six to nine samples, and bars denote SD of the mean. A, G345R and E457D; B, R320H; C, E457D titration.

 
Both G345R and E457D had strong dominant negative activity (Fig. 5AGo). Titration experiments in which different ratios of mutant and WT TRs were cotransfected suggested similar strangth of dominant negative activity by G345R and E457D (Fig. 5CGo and data not shown). These results suggest that mutant receptors that bind to DNA, but are transcriptionally inactive, can mediate this effect. The mechanism for rendering a receptor transcriptionally inactive could be due to either loss or decreased ligand binding affinity (G345R and almost all natural TRß mutants) or mutation in a putative coactivator interaction domain (E457D). In any event, the net result is the same as these receptors are unable to complex with putative coactivators such as SRC-1 when the receptors are bound to DNA. Interestingly, Takeshita et al. (17) and Collingwood et al. (36) showed weak interaction between E457D and SRC-1 in GST pull-down studies, suggesting that the protein-protein interactions may be different in solution than on DNA. In general, ligand-binding affinity of natural mutants from patients with RTH inversely correlate with dominant negative activity and suppression of TSH (1, 2, 3, 22, 37). Exceptions to this general rule may be mutants involving the AF-2 transactivation domain (21, 34, 36, 38). This region is highly conserved among nuclear hormone receptors and likely serves as a contact surface with coactivators such as SRC-1 (19, 20, 21). Recently, an extensive study of one natural mutant, L454V, showed that it had decreased interaction with SRC-1 and another putative coactivator, RIP140 (36). Interestingly, this mutant also displayed strong dominant negative activity. Our results with E457D then are in agreement with these and earlier observations with TR mutations in the AF-2 region (36, 39).

Our results with G345R and E457D also demonstrate that defective basal repression and derepression may not be the only mechanism for dominant negative activity although it may be a common feature of many natural TRß mutants (5, 35, 40). G345R had constitutive basal repression whereas E457D had normal repression and derepression; nevertheless, both displayed strong dominant negative activity. While the decreased ability to bind T3, hence release corepressor, may be a major mechanism for dominant negative activity, the data with E457D suggest that impairment of TR interaction with coactivator(s) also may be a factor mediating dominant negative activity.

Recently, several laboratories have identified the N-CoR box and adjacent sequences within the hinge region as important in mediating basal repression (7, 8, 41, 42). However, it currently is not known whether corepressor interaction itself is a prerequisite for dominant negative activity as recently suggested (35, 41). To address this issue, we made a double mutant of the N-CoR box in the hinge region (8) and the AF-2 region, hinge/457 to abrogate interactions with both corepressor and coactivator, and examined their interactions with N-CoR and SRC-1 (Fig. 6Go). However, both this mutant and the hinge mutant bind to F2 probe poorly, thus raising the possibility that weakened DNA-binding, in addition to lack of interaction with N-CoR, may account for their inability to mediate dominant negative activity. Recently, Yoh et al. (35) studied the transcriptional activity of several similar double mutants to argue that impairment of basal repression was essential for dominant negative activity, but they did not investigate DNA-binding by these mutants. We cannot rule out the possibility that hinge region mutant binding to DNA may be stabilized by interactions with other cofactors or form a complex with several proteins in vivo that were not added in our EMSA studies. Of note, though, addition of CV-1 nuclear extract was not able to stabilize hinge or hinge/457 binding to DNA (Liu, Y., and P. M. Yen, unpublished results).



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  		Figure 6. N-CoR box mutant and N-CoR box/AF-2 mutant binding to F2. In vitro translated TRß-1 and TRß-1 mutants (1 µl) containing substitutions in the N-CoR box at A223G, H224G, and T227A (8 ) (Hinge), and these substitutions and substitution at E457D (Hinge/457) were incubated with RXRß (2 µl) and N-CoR (2 µl) or GST-SRC-1 (20 ng) and [32P]-labeled F2, and then analyzed by EMSA as in Fig. 1Go. Reticulocyte lysate was added to some samples so that the total volume of reticulocyte lysate was the same for each sample. Similar amounts of TRß-1 and mutants, as quantitated by SDS-PAGE analyses, were added. 10-6 M T3 were added to some samples as indicated (Log T3 = log T3 concentration).

 
It also is interesting that R320H had weaker dominant negative activity that was further reduced when 10-6 M T3 was added. As reported previously for other mutant TRs with mild defects in ligand-binding affinity, addition of high T3 concentrations likely saturate the mutant TRs, which in turn, have normal transcriptional activity when ligand-bound, and can reverse dominant negative activity (35, 37, 43). Based on our EMSA studies in Fig. 4DGo, addition of T3 results in a larger pool of ligand-bound receptors that are competent to interact with SRC-1 or other coactivators, leading to higher transcriptional activity and reduced dominant negative activity as seen in Fig. 5BGo. We observed maximal R320H/SRC-1 formation at 10-7 M T3 on EMSA but observed increased transcriptional activity and decreased dominant negative activity by R320H in cotransfection studies at 10-6 M T3 compared with 10-7 M T3. This discrepancy likely was due to differences in the free T3 concentration, which may be 100-fold lower than the T3 concentration added to the media due to binding proteins in fetal calf sera (44). Because competition for DNA-binding between transcriptionally inactive mutant TRs and transcriptionally active WT TRs likely accounts for the amount of dominant activity observed, our studies provide a molecular mechanism by which increased circulating T3 concentration enables mutant TRs to become transcriptionally active, and thereby decrease the amount of dominant negative activity exerted by these mutant TRs.

In conclusion, we have observed impaired in vitro interactions between corepressors and coactivators, and natural mutant TRs from patients with resistance to thyroid hormone and an artificial mutant TR containing a point mutation in the AF-2 transactivation domain. The basal repression and ligand-stimulated transcription by these mutants correlates with the observed interactions with cofactors. While previous studies have demonstrated the importance of DNA-binding and dimerization on dominant negative activity by mutant TRs (4, 5, 45), the inability to interact with coactivators such as SRC-1 also can be a determinant of their dominant negative activity. Determining the precise role of coactivators on WT TR transcriptional activity will provide further details on how these mutant receptors mediate dominant negative activity.


   Acknowledgments
 
The authors would like to thank Dr. Samuel Refetoff (University of Chicago, Chicago, IL) for expression vectors encoding G345R and R320H, and Dr. Anthony Hollenberg (Beth Israel Deaconess Hospital, Boston, MA) for expression vector encoding N-CoRI.


   Footnotes
 
1 This work was supported by the March of Dimes Foundation. Back

Received November 26, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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