Specific stereochemistry of OP-1074 disrupts estrogen receptor alpha helix 12 and confers pure antiestrogenic activity.

Complex tissue-specific and cell-specific signaling by the estrogen receptor (ER) frequently leads to the development of resistance to endocrine therapy for breast cancer. Pure ER antagonists, which completely lack tissue-specific agonist activity, hold promise for preventing and treating endocrine resistance, however an absence of structural information hinders the development of novel candidates. Here we synthesize a small panel of benzopyrans with variable side chains to identify pure antiestrogens in a uterotrophic assay. We identify OP-1074 as a pure antiestrogen and a selective ER degrader (PA-SERD) that is efficacious in shrinking tumors in a tamoxifen-resistant xenograft model. Biochemical and crystal structure analyses reveal a structure activity relationship implicating the importance of a stereospecific methyl on the pyrrolidine side chain of OP-1074, particularly on helix 12.

A robust empirical seasonal prediction of winter NAO and surface climate.

A key determinant of winter weather and climate in Europe and North America is the North Atlantic Oscillation (NAO), the dominant mode of atmospheric variability in the Atlantic domain. Skilful seasonal forecasting of the surface climate in both Europe and North America is reflected largely in how accurately models can predict the NAO. Most dynamical models, however, have limited skill in seasonal forecasts of the winter NAO. A new empirical model is proposed for the seasonal forecast of the winter NAO that exhibits higher skill than current dynamical models. The empirical model provides robust and skilful prediction of the December-January-February (DJF) mean NAO index using a multiple linear regression (MLR) technique with autumn conditions of sea-ice concentration, stratospheric circulation, and sea-surface temperature. The predictability is, for the most part, derived from the relatively long persistence of sea ice in the autumn. The lower stratospheric circulation and sea-surface temperature appear to play more indirect roles through a series of feedbacks among systems driving NAO evolution. This MLR model also provides skilful seasonal outlooks of winter surface temperature and precipitation over many regions of Eurasia and eastern North America.

Atmospheric dynamics. Constrained work output of the moist atmospheric heat engine in a warming climate.

Incoming and outgoing solar radiation couple with heat exchange at Earth's surface to drive weather patterns that redistribute heat and moisture around the globe, creating an atmospheric heat engine. Here, we investigate the engine's work output using thermodynamic diagrams computed from reanalyzed observations and from a climate model simulation with anthropogenic forcing. We show that the work output is always less than that of an equivalent Carnot cycle and that it is constrained by the power necessary to maintain the hydrological cycle. In the climate simulation, the hydrological cycle increases more rapidly than the equivalent Carnot cycle. We conclude that the intensification of the hydrological cycle in warmer climates might limit the heat engine's ability to generate work.

A splice variant of estrogen receptor beta missing exon 3 displays altered subnuclear localization and capacity for transcriptional activation.

There are two separate estrogen receptors (ERs), ERalpha and ERbeta. The ERbeta gene is variably spliced, and in some cases variant expression is high. Besides the full-length ERbeta (equivalent to ERbeta1), splice variants can encode proteins bearing an insert within the ligand-binding domain (beta2), a deletion of exon 3 (ERbeta1delta3) disrupting the DNA-binding domain, or both (ERbeta2delta3). Here we examine the intracellular localization and transcriptional properties of each of the ERbeta splice variants heterologously expressed in cultured cells. In accordance with ERalpha, ERbeta1 and ERbeta2 are both distributed in a reticular pattern within the nucleus after exposure to ligand. In contrast, ERbeta1delta3 and ERbeta2delta3 localize to discrete spots within the nucleus in the presence of ER agonists. In the presence of ER antagonists, the delta3 variants are distributed diffusely within the nucleus. We also show that the spots are stable nuclear structures to which the delta3 variants localize in a ligand-dependent manner. Coactivator proteins of ER colocalize with delta3 variants in the spots in the presence of agonists. The delta3 variants of ERbeta can activate luciferase reporter constructs containing an activator protein complex-1 site, but not an estrogen response element (ERE). These data suggest that without an intact DNA-binding domain, ERbeta is functionally altered, allowing localization to discrete nuclear spots and activation from activator protein-1-containing reporter genes.

Growth factors signal to steroid receptors through mitogen-activated protein kinase regulation of p160 coactivator activity.

Promoter-bound steroid receptors activate gene expression by recruiting members of the p160 family of coactivators. Many steroid receptors, most notably the progesterone and estrogen receptors, are regulated both by cognate hormone and independently by growth factors. Here we show that epidermal growth factor regulates the activities of the p160 GRIP1 through the extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinases. ERKs phosphorylate GRIP1 at a specific site, Ser-736, the integrity of which is required for full growth factor induction of GRIP1 transcriptional activation and coactivator function. We propose that growth factors signal to nuclear receptors in part by targeting the p160 coactivators.

Role of direct interaction in BRCA1 inhibition of estrogen receptor activity.

The BRCA1 gene was previously found to inhibit the transcriptional activity of the estrogen receptor [ER-alpha] in human breast and prostate cancer cell lines. In this study, we found that breast cancer-associated mutations of BRCA1 abolish or reduce its ability to inhibit ER-alpha activity and that domains within the amino- and carboxyl-termini of the BRCA1 protein are required for the inhibition. BRCA1 inhibition of ER-alpha activity was demonstrated under conditions in which a BRCA1 transgene was transiently or stably over-expressed in cell lines with endogenous wild-type BRCA1 and in a breast cancer cell line that lacks endogenous functional BRCA1 (HCC1937). In addition, BRCA1 blocked the expression of two endogenous estrogen-regulated gene products in human breast cancer cells: pS2 and cathepsin D. The BRCA1 protein was found to associate with ER-alpha in vivo and to bind to ER-alpha in vitro, by an estrogen-independent interaction that mapped to the amino-terminal region of BRCA1 (ca. amino acid 1-300) and the conserved carboxyl-terminal activation function [AF-2] domain of ER-alpha. Furthermore, several truncated BRCA1 proteins containing the amino-terminal ER-alpha binding region blocked the ability of the full-length BRCA1 protein to inhibit ER-alpha activity. Our findings suggest that the amino-terminus of BRCA1 interacts with ER-alpha, while the carboxyl-terminus of BRCA1 may function as a transcriptional repression domain. Oncogene (2001) 20, 77 - 87.

Direct acetylation of the estrogen receptor alpha hinge region by p300 regulates transactivation and hormone sensitivity.

Regulation of nuclear receptor gene expression involves dynamic and coordinated interactions with histone acetyl transferase (HAT) and deacetylase complexes. The estrogen receptor (ERalpha) contains two transactivation domains regulating ligand-independent and -dependent gene transcription (AF-1 and AF-2 (activation functions 1 and 2)). ERalpha-regulated gene expression involves interactions with cointegrators (e.g. p300/CBP, P/CAF) that have the capacity to modify core histone acetyl groups. Here we show that the ERalpha is acetylated in vivo. p300, but not P/CAF, selectively and directly acetylated the ERalpha at lysine residues within the ERalpha hinge/ligand binding domain. Substitution of these residues with charged or polar residues dramatically enhanced ERalpha hormone sensitivity without affecting induction by MAPK signaling, suggesting that direct ERalpha acetylation normally suppresses ligand sensitivity. These ERalpha lysine residues also regulated transcriptional activation by histone deacetylase inhibitors and p300. The conservation of the ERalpha acetylation motif in a phylogenetic subset of nuclear receptors suggests that direct acetylation of nuclear receptors may contribute to additional signaling pathways involved in metabolism and development.

Potentiation of estrogen receptor activation function 1 (AF-1) by Src/JNK through a serine 118-independent pathway.

Estrogen receptor (ER) is activated either by ligand or by signals from tyrosine kinase-linked cell surface receptors. We investigated whether the nonreceptor Src tyrosine kinase could affect ER activity. Expression of constitutively active Src or stimulation of the endogenous Src/JNK pathway enhances transcriptional activation by the estrogen-ER complex and strongly stimulates the otherwise weak activation by the unliganded ER and the tamoxifen-ER complex. Src affects ER activation function 1 (AF-1), and not ER AF-2, and does so through its tyrosine kinase activity. This effect of Src is mediated partly through a Raf/mitogen-activated ERK kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) signaling cascade and partly through a MEKK/JNKK/JNK cascade. Although, as previously shown, Src action through activated ERK stimulates AF-1 by phosphorylation at S118, Src action through activated JNK neither leads to phosphorylation of S118 nor requires S118 for its action. We therefore suggest that the Src/JNK pathway enhances AF-1 activity by modification of ER AF-1-associated proteins. Src potentiates activation functions in CREB-binding protein (CBP) and glucocorticoid receptor interacting protein 1 (GRIP1), and we discuss the possibility that the Src/JNK pathway enhances the activity of these coactivators, which are known to mediate AF-1 action.

The nuclear receptor corepressor (N-CoR) contains three isoleucine motifs (I/LXXII) that serve as receptor interaction domains (IDs).

Unliganded thyroid hormone receptors (TRs) repress transcription through recruitment of corepressors, including nuclear receptor corepressor (N-CoR). We find that N-CoR contains three interaction domains (IDs) that bind to TR, rather than the previously reported two. The hitherto unrecognized ID (ID3) serves as a fully functional TR binding site, both in vivo and in vitro, and may be the most important for TR binding. Each ID motif contains a conserved hydrophobic core (I/LXXII) that resembles the hydrophobic core of nuclear receptor boxes (LXXLL), which mediates p160 coactivator binding to liganded nuclear receptors. Although the integrity of the I/LXXII motif is required for ID function, substitution of ID isoleucines with leucines did not allow ID peptides to bind to liganded TR, and substitution of NR box leucines with isoleucines did not allow NR box peptides to bind unliganded TR. This indicates that the binding preferences of N-CoR for unliganded TR and p160s for liganded TR are not dictated solely by the identity of conserved hydrophobic residues within their TR binding motifs. Examination of sequence conservation between IDs, and mutational analysis of individual IDs, suggests that they are comprised of the central hydrophobic core and distinct adjacent sequences that may make unique contacts with the TR surface. Accordingly, a hybrid peptide that contains distinct adjacent sequences from ID3 and ID1 shows enhanced binding to TR.

An antiestrogen-responsive estrogen receptor-alpha mutant (D351Y) shows weak AF-2 activity in the presence of tamoxifen.

Antiestrogens, including tamoxifen and raloxifene, block estrogen receptor (ER) action by blocking the interactions of an estrogen-dependent activation function (AF-2) with p160 coactivators. Although tamoxifen does show some agonist activity in the presence of ERalpha, this stems from a distinct constitutive activation function (AF-1) that lies within the ERalpha N terminus. Previous studies identified a naturally occurring mutation (D351Y) that allows ERalpha to perceive tamoxifen and raloxifene as estrogens. Here, we examine the contributions of ERalpha activation functions to the D351Y phenotype. We find that the AF-2 function of ERalpha D351Y lacks detectable tamoxifen-dependent activity when tested in isolation but does synergize with AF-1 to allow enhanced tamoxifen response. Weak tamoxifen-dependent interactions between the ERalpha D351Y AF-2 function and GRIP1, a representative p160, can be detected in glutathione S-transferase binding assays and mammalian two-hybrid assays. Furthermore, tamoxifen-dependent AF-2 activity can be detected in the presence of ERalpha D351Y and high levels of overexpressed GRIP1. We therefore propose that the D351Y mutation allows weak tamoxifen-dependent AF-2 activity but that this activity is only detectable when AF-1 is strong, and AF-1 and AF-2 synergize, or when p160s are overexpressed. We discuss the possible structural basis of this effect.

Dynamic stabilization of nuclear receptor ligand binding domains by hormone or corepressor binding.

We have developed a novel assembly assay to examine structural changes in the ligand binding domain (LBD) of the thyroid hormone receptor (TR). Fragments including the first helix of the TR LBD interact only weakly with the remainder of the LBD in the absence of hormone, but this interaction is strongly enhanced by the addition of either hormone or the corepressor NCoR. Since neither the ligand nor the corepressor shows direct interaction with this helix, we propose that both exert their effects by stabilizing the overall structure of the LBD. Current models of activation of nuclear hormone receptors focus on a ligand-induced allosteric shift in the position of the C-terminal helix 12 that generates the coactivator binding site. Our results suggest that ligand binding also has more global effects that dynamically alter the structure of the receptor LBD.

Oestrogen receptor function at classical and alternative response elements.

The oestrogen receptor (ER), bound to classical response elements (EREs) in the promoter of target genes, activates transcription by recruiting coactivator proteins. We will describe structural studies that show that oestrogens allow the formation of a hydrophobic cleft on the surface of the ER that serves as a docking site for coactivators. Anti-oestrogens displace part of the receptor, which then occludes the site, blocking coactivator access. In addition to activating at classical EREs, the ER activates transcription at alternative elements such as AP-1 sites. These bind the Jun/Fos proteins but not ER. Interestingly both oestrogen and tamoxifen activate transcription at AP-1 sites. We propose a mechanism whereby oestrogen and anti-oestrogen allow ER to activate transcription from alternative response elements. ER binds to the coactivators, CBP and GRIP1, that have been recruited by Jun/Fos and through this contact 'triggers' these coactivators into full activity. In this circumstance the ER is part of the coactivator complex for Jun/Fos.

Estrogen receptor pathways to AP-1.

Estrogen receptor (ER) binds to estrogen response elements in target genes and recruits a coactivator complex of CBP-pl60 that mediates stimulation of transcription. ER also activates transcription at AP-1 sites that bind the Jun/Fos transcription factors, but not ER. We review the evidence regarding mechanisms whereby ER increases the activity of Jun/Fos and propose two pathways of ER action depending on the ER (alpha or beta) and on the ligand. We propose that estrogen-ERalpha complexes use their activation functions (AF-1 and AF-2) to bind to the p 160 component of the coactivator complex recruited by Jun/Fos and trigger the coactivator to a higher state of activity. We propose that selective estrogen receptor modulator (SERM) complexes with ERbeta and with truncated ERalpha derivatives use their DNA binding domain to titrate histone deacetylase (HDAC)-repressor complexes away from the Jun/Fos coactivator complex, thereby allowing unfettered activity of the coactivators. Finally, we consider the possible physiological significance of ER action at AP-1 sites.

Estradiol repression of tumor necrosis factor-alpha transcription requires estrogen receptor activation function-2 and is enhanced by coactivators.

The tumor necrosis factor-alpha (TNF-alpha) promoter was used to explore the molecular mechanisms of estradiol (E(2))-dependent repression of gene transcription. E(2) inhibited basal activity and abolished TNF-alpha activation of the TNF-alpha promoter. The E(2)-inhibitory element was mapped to the -125 to -82 region of the TNF-alpha promoter, known as the TNF-responsive element (TNF-RE). An AP-1-like site in the TNF-RE is essential for repression activity. Estrogen receptor (ER) beta is more potent than ERalpha at repressing the -1044 TNF-alpha promoter and the TNF-RE upstream of the herpes simplex virus thymidine kinase promoter, but weaker at activating transcription through an estrogen response element. The activation function-2 (AF-2) surface in the ligand-binding domain is required for repression, because anti-estrogens and AF-2 mutations impair repression. The requirement of the AF-2 surface for repression is probably due to its capacity to recruit p160 coactivators or related coregulators, because overexpressing the coactivator glucocorticoid receptor interacting protein-1 enhances repression, whereas a glucocorticoid receptor interacting protein-1 mutant unable to interact with the AF-2 surface is ineffective. Furthermore, receptor interacting protein 140 prevents repression by ERbeta, probably by interacting with the AF-2 surface and blocking the binding of endogenous coactivators. These studies demonstrate that E(2)-mediated repression requires the AF-2 surface and the participation of coactivators or other coregulatory proteins.

The estrogen receptor enhances AP-1 activity by two distinct mechanisms with different requirements for receptor transactivation functions.

Estrogen receptors (ERs alpha and beta) enhance transcription in response to estrogens by binding to estrogen response elements (EREs) within target genes and utilizing transactivation functions (AF-1 and AF-2) to recruit p160 coactivator proteins. The ERs also enhance transcription in response to estrogens and antiestrogens by modulating the activity of the AP-1 protein complex. Here, we examine the role of AF-1 and AF-2 in ER action at AP-1 sites. Estrogen responses at AP-1 sites require the integrity of the ERalpha AF-1 and AF-2 activation surfaces and the complementary surfaces on the p160 coactivator GRIP1 (glucocorticoid receptor interacting protein 1), the NID/AF-1 region, and NR boxes. Thus, estrogen-liganded ERalpha utilizes the same protein-protein contacts to transactivate at EREs and AP-1 sites. In contrast, antiestrogen responses are strongly inhibited by ERalpha AF-1 and weakly inhibited by AF-2. Indeed, ERalpha truncations that lack AF-1 enhance AP-1 activity in the presence of antiestrogens, but not estrogens. This phenotype resembles ERbeta, which naturally lacks constitutive AF-1 activity. We conclude that the ERs enhance AP-1 responsive transcription by distinct mechanisms with different requirements for ER transactivation functions. We suggest that estrogen-liganded ER enhances AP-1 activity via interactions with p160s and speculate that antiestrogen-liganded ER enhances AP-1 activity via interactions with corepressors.

Multiple signal input and output domains of the 160-kilodalton nuclear receptor coactivator proteins.

Members of the 160-kDa nuclear receptor coactivator family (p160 coactivators) bind to the conserved AF-2 activation function found in the hormone binding domains of nuclear receptors (NR) and are potent transcriptional coactivators for NRs. Here we report that the C-terminal region of p160 coactivators glucocorticoid receptor interacting protein 1 (GRIP1), steroid receptor coactivator 1 (SRC-1a), and SRC-1e binds the N-terminal AF-1 activation function of the androgen receptor (AR), and p160 coactivators can thereby enhance transcriptional activation by AR. While they all interact efficiently with AR AF-1, these same coactivators have vastly different binding strengths with and coactivator effects on AR AF-2. p160 activation domain AD1, which binds secondary coactivators CREB binding protein (CBP) and p300, was previously implicated as the principal domain for transmitting the activating signal to the transcription machinery. We identified a new highly conserved motif in the AD1 region which is important for CBP/p300 binding. Deletion of AD1 only partially reduced p160 coactivator function, due to signaling through AD2, another activation domain located at the C-terminal end of p160 coactivators. C-terminal coactivator fragments lacking AD1 but containing AD2 and the AR AF-1 binding site served as efficient coactivators for full-length AR and AR AF-1. The two signal input domains (one that binds NR AF-2 domains and one that binds AF-1 domains of some but not all NRs) and the two signal output domains (AD1 and AD2) of p160 coactivators played different relative roles for two different NRs: AR and thyroid hormone receptor.

Titration by estrogen receptor activation function-2 of targets that are downstream from coactivators.

Cross-interference (squelching) among nuclear receptors has been proposed to reflect the titration of coactivators that bind the receptors in a hormone-dependent manner. We have tested whether the coactivators are the only target titrated during squelching of one receptor by another, or whether proteins needed for coactivator function are titrated as well. That the coactivators are indeed one target of squelching is apparent. The isolated ligand-binding domain of the estrogen receptor (ER-LBD) squelches transcriptional activation by the thyroid hormone receptor (TR) only when the LBD is bound to ligands that promote coactivator interactions and only when regions of the LBD that promote coactivator interactions are undisturbed. Furthermore, the ER-LBD and the TR compete in vitro for the related p160 coactivators, SRC1a and GRIP1 (glucocorticoid receptor interacting protein 1), or the putative corepressor, RIP140. Finally TR action becomes more potent when coactivator levels are raised. Nonetheless, supplying excess SRC1a or GRIP1 does not abolish squelching by the ER. In fact, squelching becomes even more severe when coactivators are abundant. Supplying combinations of coactivators from the p160 class and the CREB-binding protein (CBP)/p300 class makes squelching most severe. Elevated RIP140 inhibits TR action, but also protects the residual TR action from squelching by the ER-LBD. We conclude that ER-LBD squelches TR both by titrating p160-CBP coactivators and additionally by cooperating with the coactivators to titrate a second factor. The second factor would be needed by the TR for coactivator-mediated transcriptional stimulation.

Increased activator protein-1 DNA binding and c-Jun NH2-terminal kinase activity in human breast tumors with acquired tamoxifen resistance.

Human breast tumors that are initially responsive to tamoxifen (TAM) eventually relapse during treatment. Estrogen receptor (ER) expression and function are often preserved in these tumors, and clinical evidence suggests that this relapse may be related to TAM's known agonistic properties. ER can interact with the activator protein-1 (AP-1) transcription factor complex through protein-protein interactions that are independent of ER DNA binding and, in certain ER-positive cells, this may allow TAM to exert an agonist response on AP-1-regulated genes. We, therefore, assessed both AP-1 DNA binding and the known AP-1 activating enzyme, c-Jun NH2-terminal kinase (JNK), in a panel of 30 ER-positive primary human breast tumors with acquired TAM resistance, as compared to a matched panel of 27 untreated control ER-positive breast tumors and a separate control set of 14 primary tumors, which included 7 ER-positive tumors that were growth-arrested by 3 months of preoperative TAM. AP-1 DNA binding activity was measured from cryopreserved tumor extracts using a labeled oligonucleotide probe containing a consensus AP-1 response element by electrophoretic mobility shift assay. JNK was first extracted from the tumor lysates by incubation over a Sepharose-bound c-Jun(1-89) fusion protein, and its activity was then measured by chemiluminescent Western blot by detection of the phosphorylated product using a phospho-Jun(Ser-63)-specific primary antibody. The set of control ER-positive breast tumors growth arrested by TAM showed no significant difference from untreated control tumors in their AP-1 DNA binding and JNK activities. In contrast, there was a significant (P < 0.001) increase in mean AP-1 DNA binding activity for the panel of ER-positive TAM-resistant (TAM-R) tumors as compared to its matched control panel of untreated tumors. Mean JNK activity in the TAM-R tumors was also significantly higher than that found in the untreated tumors (P = 0.038). Overall, there was no significant correlation between JNK activity and AP-1 DNA binding; however, regression analysis showed that, for any given level of JNK activity, the TAM-R tumors possessed a 3.5-fold increase in AP-1 DNA binding activity as compared to the untreated tumors. These findings indicate that, when compared to untreated ER-positive primary breast tumors, TAM-R tumors demonstrate significantly increased levels of AP-1 DNA binding and JNK activity, consistent with experimental models suggesting that TAM-stimulated ER-positive tumor growth may be mediated by enhanced AP-1 transcriptional activity. These observations support the need for further evaluation of these markers in breast tumors as predictors of TAM resistance.

Molecular and structural biology of thyroid hormone receptors.

1. Thyroid hormone receptors (TR) are expressed from two separate genes (alpha and beta) and belong to the nuclear receptor superfamily, which also contains receptors for steroids, vitamins and prostaglandins. 2. Unliganded TR are bound to DNA thyroid hormone response elements (TRE) predominantly as homodimers, or as heterodimers with retinoid X-receptors (RXR), and are associated with a complex of proteins containing corepressor proteins. Ligand binding promotes corepressor dissociation and binding of a coactivator. 3. Recent studies from our group have focused on the acquisition and use of X-ray crystallographic structures of ligand-binding domains (LBD) of both the rat (r) TR alpha and the human (h) TR beta bound to several different ligands. We have also developed ligands that bind selectively to the TR beta, which may provide ways to explore the differential functions of TR alpha compared with TR beta isoforms. 4. The LBD is comprised mostly of alpha-helices. The ligand is completely buried in the receptor and forms part of its hydrophobic core. Kinetic studies suggest that the limiting step in formation of high-affinity ligand-receptor complexes is the rate of folding of the receptor around the ligand. Ligands can be fitted tightly in the ligand-binding pocket and small differences in this fitting may explain many structure-activity relationships. Interestingly, analysis of the structures of antagonists suggests that they have chemical groups, 'extensions', that could impair receptor folding around them and, thus, prevent the agonist-induced conformation changes in the receptor. 5. The TR structures allowed us to see that the mutations that occur in the syndrome of generalized resistance to thyroid hormone are located in the vicinity of the ligand-binding pocket. 6. X-ray structure of the TR has also been used to guide construction of mutations in the TR surface that block binding of various proteins important for receptor function. Studies with these TR mutants reveal that the interfaces for homo- and heterodimerization map to similar residues in helix 10 and 11 and also allow the definition of the surface for binding of coactivators, which appears to be general for nuclear receptors. Formation of this surface, which involves packing of helix 12 of the TR into a scaffold formed by helices 3 and 5, appears to be the major change in the receptor structure induced by hormone occupancy.

Structure and specificity of nuclear receptor-coactivator interactions.

Combinatorial regulation of transcription implies flexible yet precise assembly of multiprotein regulatory complexes in response to signals. Biochemical and crystallographic analyses revealed that hormone binding leads to the formation of a hydrophobic groove within the ligand binding domain (LBD) of the thyroid hormone receptor that interacts with an LxxLL motif-containing alpha-helix from GRIP1, a coactivator. Residues immediately adjacent to the motif modulate the affinity of the interaction; the motif and the adjacent sequences are employed to different extents in binding to different receptors. Such interactions of amphipathic alpha-helices with hydrophobic grooves define protein interfaces in other regulatory complexes as well. We suggest that these common structural elements impart flexibility to combinatorial regulation, whereas side chains at the interface impart specificity.