Control of glucocorticoid and progesterone receptor subcellular localization by the ligand-binding domain is mediated by distinct interactions with tetratricopeptide repeat proteins

The TPR proteins FKBP52, FKBP51, Cyp40, and PP5 are found in steroid receptor (SR) complexes, but their receptor-specific preferences and roles remain unresolved. We have undertaken a systematic approach to this problem by examining the contribution of all four TPRs to the localization properties of glucocorticoid (GR) and progesterone (PR) receptors. The GR of L929 cells was found in the cytoplasm in a complex containing PP5 and FKBP51, while the GR of WCL2 cells was nuclear and contained PP5 and FKBP52. Cyp40 did not interact with the GR in either cell line. To test whether FKBP interaction determined localization, we overexpressed Flag-tagged FKBP51 in WCL2 cells and Flag-FKBP52 in L929 cells. In WCL2 cells, the GR exhibited a shift to greater cytoplasmic localization that correlated with recruitment of Flag-FKBP51. In contrast, Flag-FKBP52 was not recruited to the GR of L929 cells, and no change in localization was observed, suggesting that both cell-type-specific mechanisms and TPR abundance contribute to the SR-TPR interaction. As a further test, GR-GFP and PR-GFP constructs were expressed in COS cells. The GR-GFP construct localized to the cytoplasm, while the PR-GFP construct was predominantly nuclear. Similar to L929 cells, the GR in COS interacted with PP5 and FKBP51, while PR interacted with FKBP52. Analysis of GR-PR chimeric constructs revealed that the ligand-binding domain of each receptor determines both TPR specificity and localization. Lastly, we analyzed GR and PR localization in cells completely lacking TPR. PR in FKBP52 KO cells showed a complete shift to the cytoplasm, while GR in FKBP51 KO and PP5 KO cells showed a moderate shift to the nucleus, indicating that both TPRs contribute to GR localization. Our results demonstrate that SRs have distinct preferences for TPR proteins, a property that resides in the LBD and which can now explain long-standing differences in receptor subcellular localization.     

Folding and stability of the ligand-binding domain of the glucocorticoid receptor

A complex pathway involving many molecular chaperones has been proposed for the folding, assembly, and maintenance of a high-affinity ligand-binding form of steroid receptors in vivo, including the glucocorticoid receptor. To better understand this intricate folding and assembly process, we studied the folding of the ligand-binding domain of the glucocorticoid receptor in vitro. We found that this domain can be refolded into a compact, highly structured state in vitro in the absence of chaperones. However, the presence of zwitterionic detergent is required to maintain the domain in a soluble form. In this state, the protein is dimeric and has considerable helical structure as shown by far-UV circular dichroism. Further investigation of the properties of this in vitro refolded state show that it is stable and resistant to denaturation by heat or low concentrations of chemical denaturants. A detailed analysis of the unfolding equilibria using three different structural probes demonstrated that this state unfolds via a highly populated dimeric intermediate state. Together, these data clearly show that the ligand-binding domain of the glucocorticoid receptor does not require chaperones for folding per se. However, this in vitro refolded state binds the ligand dexamethasone only weakly (K(d) = 45 microM) compared to the in vivo assembled receptor (K(d) = 3.4 nM). We suggest that the role of Hsp90 and associated chaperones is to bind to, and stabilize, a specific conformational state of the receptor which binds ligand with high affinity.     

Noncontiguous regions in the extracellular domain of EGF receptor define ligand-binding specificity.

Murine epidermal growth factor (EGF) binds with approximately 250-fold higher binding affinity to the human EGF receptor (EGFR) than to the chicken EGFR. This difference in binding affinity enabled the identification of a major ligand-binding domain for EGF by studying the binding properties of various chicken/human EGFR chimera expressed in transfected cells lacking endogenous EGFR. It was shown that domain III of EGFR is a major ligand-binding region. Here, we analyze the binding properties of novel chicken/human chimera to further delineate the contact sequences in domain III and to assess the role of other regions of EGFR for their contribution to the display of high-affinity EGF binding. The chimeric receptors include chicken EGFR containing domain I of the human EGFR, chicken receptor containing domain I and III of the human EGFR, and two chimeric chicken EGFR containing either the amino terminal or the carboxy terminal halves of domain III of human EGFR, respectively. In addition, the binding of various human-specific anti-EGFR monoclonal antibodies that interfere with EGF binding is also compared. It is concluded that noncontiguous regions of the EGFR contribute additively to the binding of EGF. Each of the two halves of domain III has a similar contribution to the binding energy, and the sum of both is close to that of the entire domain III. This suggests that the folding of domain III juxtaposes sequences that together constitute the ligand-binding site. Domain I also provides a contribution to the binding energy, and the added contributions of both domain I and III to the binding energy generate the high-affinity binding site typical of human EGFR.     

Structural features of the ligand-binding domain of the serotonin 5HT3 receptor

The nicotinic acetylcholine receptor (AChR) and the serotonin type 3 receptor (5HT3R) are members of the ligand-gated ion channel gene family. Both receptors are inhibited by nanomolar concentrations of d-tubocurarine (curare) in a competitive fashion. Chemical labeling studies on the AChR have identified tryptophan residues on the gamma (gammaTrp-55) and delta (deltaTrp-57) subunits that interact with curare. Comparison of the sequences of these two subunits with the 5HT3R shows that a tryptophan residue is found in the homologous position in the 5HT3R (Trp-89), suggesting that this residue may be involved in curare-5HT3R interactions. Site-directed mutagenesis at position Trp-89 markedly reduces the affinity of the 5HT3R for the antagonists curare and granisetron but has little effect on the affinity for the agonist serotonin. To further examine the role of this region of the receptor in ligand-receptor interactions, alanine-scanning mutagenesis analysis of the region centered on Trp-89 (Thr-85 to Trp-94) was carried out, and the ligand binding properties of the mutant receptors were determined. Within this region of the receptor, curare affinity is reduced by substitution only at Trp-89, whereas serotonin affinity is reduced only by substitution at Arg-91. On the other hand, granisetron affinity is reduced by substitutions at Trp-89, Arg-91, and Tyr-93. This differential effect of substitutions on ligand affinity suggests that different ligands may have different points of interaction within the ligand-binding pocket. In addition, the every-other-residue periodicity of the effects on granisetron affinity strongly suggests that this region of the ligand-binding site of the 5HT3R (and by inference, other members of the ligand-gated ion channel family) is in a beta-strand conformation.     

Classical Xenopus laevis progesterone receptor associates to the plasma membrane through its ligand-binding domain

During the last decade, considerable evidence is accumulating that supports the view that the classic progesterone receptor (xPR-1) is mediating Xenopus laevis oocyte maturation through a non-genomic mechanism. Overexpression and depletion of oocyte xPR-1 have been shown to accelerate and to block progesterone-induced oocyte maturation, respectively. In addition, rapid inhibition of plasma membrane adenylyl cyclase (AC) by the steroid hormone, supports the idea that xPR-1 should be localized at the oocyte plasma membrane. To test this hypothesis, we transiently transfected xPR-1 cDNA into Cos-7 cells and analyzed its subcellular distribution. Through Western blot and immunofluorescence analysis, we were able to detect xPR-1 associated to the plasma membrane of transfected Cos-7 cells. Additionally, using Progesterone-BSA-FITC, we identified specific progesterone-binding sites at the cell surface of xPR-1 expressing cells. Finally, we found that the receptor ligand-binding domain displayed membrane localization, in contrast to the N-terminal domain, which expressed in similar levels, remained cytosolic. Overall, these results indicate that a fraction of xPR-1 expressed in Cos-7 cells, associates to the plasma membrane through its LBD.     

Cooperativity and specificity in the interactions between DNA and the glucocorticoid receptor DNA-binding domain

We have employed fluorescence spectroscopy to study the chemical equilibrium between a 115 amino acid protein fragment containing the DNA-binding domain of the human glucocorticoid receptor (DBDr) and a 24-base-pair DNA oligomer containing the glucocorticoid response element (GRE) from the mouse mammary tumor virus promoter region and compared it with the binding to nonspecific DNA at various ionic conditions. We find that binding to both DNAs is cooperative but that DBDr shows a higher affinity for the GRE than for nonspecific DNA and that this difference is more pronounced at increased salt concentrations. Sequence-specific binding to the GRE sequence at 570 mM monovalent cations can be described by a two-site cooperative model, and this supports the notion that DBDr binding to the GRE is enhanced by dimer formation at the recognition site. The product between the (average) association constant for binding to a GRE half-site and the cooperativity parameter was estimated to be K omega = (1-4) x 10(7) M-1 at this salt concentration and 20 degrees C. The sequence-specific binding is not very sensitive to salt concentration in the interval 270-570 mM monovalent cations. However, at lower salt (70 mM) additional binding takes place, presumably nonspecific (cooperative) association to DNA adjacent to the GRE sequence. DBDr binding to nonspecific DNA can be described by the McGhee-von Hippel model for cooperative binding to a chain polymer and is very sensitive to ionic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular modeling of the GABAC receptor ligand-binding domain

We have constructed a molecular model of the ligand-binding domain of the GABA(C) receptor, which is a member of the Cys-loop ligand-gated ion channel family. The extracellular domains of these receptors share similar sequence homology (20%) with Limnaea acetylcholine-binding protein for which an X-ray crystal structure is available. We used this structure as a template for homology modeling of the GABA(C) receptor extracellular domain using FUGUE and MODELLER software. FlexX was then used to dock GABA into the receptor ligand-binding site, resulting in three alternative energetically favorable orientations. Residues located no more than 5 A from the docked GABA were identified for each model; of these, three were found to be common to all models with 14 others present only in certain models. Using data from experimental studies, we propose that the most likely orientation of GABA is with its amine close to Y198, and its carboxylate close to R104. These studies have therefore provided a model of the ligand-binding domain, which will be useful for both GABA(C) and GABA(A) receptor studies, and have also yielded an experimentally testable hypothesis of the location of GABA in the binding pocket. [Figure: see text].     

Structural plasticity in the oestrogen receptor ligand-binding domain

The steroid hormone receptors are characterized by binding to relatively rigid, inflexible endogenous steroid ligands. Other members of the nuclear receptor superfamily bind to conformationally flexible lipids and show a corresponding degree of elasticity in the ligand-binding pocket. Here, we report the X-ray crystal structure of the oestrogen receptor alpha (ERalpha) bound to an oestradiol derivative with a prosthetic group, ortho- trifluoromethlyphenylvinyl, which binds in a novel extended pocket in the ligand-binding domain. Unlike ER antagonists with bulky side groups, this derivative is enclosed in the ligand-binding pocket, and acts as a potent agonist. This work shows that steroid hormone receptors can interact with a wider array of pharmacophores than previously thought through structural plasticity in the ligand-binding pocket.     

Stargazin promotes closure of the AMPA receptor ligand-binding domain

Transmembrane AMPA receptor (AMPAR) regulatory proteins (TARPs) markedly enhance AMPAR function, altering ligand efficacy and receptor gating kinetics and thereby shaping the postsynaptic response. The structural mechanism underlying TARP effects on gating, however, is unknown. Here we find that the prototypical member of the TARP family, stargazin or γ-2, rescues gating deficits in AMPARs carrying mutations that destabilize the closed-cleft states of the ligand-binding domain (LBD), suggesting that stargazin reverses the effects of these mutations and likely stabilizes closed LBD states. Furthermore, stargazin promotes a more closed conformation of the LBD, as indicated by reduced accessibility to the large antagonist NBQX. Consistent with the functional studies, luminescence resonance energy transfer experiments directly demonstrate that the AMPAR LBD is on average more closed in the presence of stargazin, in both the apo and agonist-bound states. The additional cleft closure and/or stabilization of the more closed-cleft states of the LBD is expected to translate to higher agonist efficacy and could contribute to the structural mechanism for stargazin modulation of AMPAR function.     

Mutational analysis of the ligand-binding domain of the prolactin receptor

The recent isolation and sequencing of the rat liver prolactin (PRL) receptor cDNA (clone F3) revealed that the receptor is a small molecular weight protein (nonglycosylated form, Mr 33,000; glycosylated form, Mr 42,000) comprised of 291 amino acids. A second form of the PRL receptor exists (591 amino acids) that contains a much longer cytoplasmic domain. In the present study, site-directed point mutations of the 5 conserved cysteine (Cys) residues and of the three potential N-linked glycosylation sites in the extracellular domain of the rat PRL receptor were constructed to assess their involvement in hormone binding. In addition, a truncation mutant (T delta 237) lacking 55 of 57 intracellular amino acids was constructed to determine the influence of the cytoplasmic domain on ligand-receptor interactions. Binding studies of transiently transfected COS-7 cells demonstrated that serine substitution of the first 4 Cys residues (Cys12, Cys22, Cys51, and Cys62) completely eliminated binding of 125I-ovine PRL and 125I-U5 and -U6, two monoclonal antibodies that bind the receptor molecule outside the PRL-binding domain. RNA blot analysis of the transfected cells showed that both the wild-type and mutant clones had similar levels of expression of receptor mRNA. Immunoblot analysis demonstrated that lack of PRL binding in these mutants was not due to incomplete processing of the protein, since the fully glycosylated Mr 42,000 form of the receptor was seen. Mutation of Cys184 had no effect on affinity or dimerization capacity of the receptor, suggesting the 5th cysteine is not directly involved in the binding domain. Carbohydrate groups of some receptors have been shown to be involved in ligand-receptor interactions as well as intracellular trafficking. This does not appear to be the case for the PRL receptor, since there was no corresponding decrease in affinity for PRL or cell surface receptor expression, following mutation of each of the 3 asparagine residues to aspartate. Interestingly, T delta 237 showed a 4-5-fold increase in affinity for PRL as well as a marked increase in the number of receptor sites. Whole cell binding assays also demonstrated that loss of the cytoplasmic domain lead to inefficient recycling of the receptor. These studies suggest that the first 4 conserved Cys residues are crucial for ligand binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Allosteric conversation in the androgen receptor ligand-binding domain surfaces

Androgen receptor (AR) is a major therapeutic target that plays pivotal roles in prostate cancer (PCa) and androgen insensitivity syndromes. We previously proposed that compounds recruited to ligand-binding domain (LBD) surfaces could regulate AR activity in hormone-refractory PCa and discovered several surface modulators of AR function. Surprisingly, the most effective compounds bound preferentially to a surface of unknown function [binding function 3 (BF-3)] instead of the coactivator-binding site [activation function 2 (AF-2)]. Different BF-3 mutations have been identified in PCa or androgen insensitivity syndrome patients, and they can strongly affect AR activity. Further, comparison of AR x-ray structures with and without bound ligands at BF-3 and AF-2 showed structural coupling between both pockets. Here, we combine experimental evidence and molecular dynamic simulations to investigate whether BF-3 mutations affect AR LBD function and dynamics possibly via allosteric conversation between surface sites. Our data indicate that AF-2 conformation is indeed closely coupled to BF-3 and provide mechanistic proof of their structural interconnection. BF-3 mutations may function as allosteric elicitors, probably shifting the AR LBD conformational ensemble toward conformations that alter AF-2 propensity to reorganize into subpockets that accommodate N-terminal domain and coactivator peptides. The induced conformation may result in either increased or decreased AR activity. Activating BF-3 mutations also favor the formation of another pocket (BF-4) in the vicinity of AF-2 and BF-3, which we also previously identified as a hot spot for a small compound. We discuss the possibility that BF-3 may be a protein-docking site that binds to the N-terminal domain and corepressors. AR surface sites are attractive pharmacological targets to develop allosteric modulators that might be alternative lead compounds for drug design.     

Androgen receptor ligand-binding domain interaction and nuclear receptor specificity of FXXLF and LXXLL motifs as determined by L/F swapping

The androgen receptor (AR) ligand-binding domain (LBD) binds FXXLF motifs, present in the AR N-terminal domain and AR-specific cofactors, and some LXXLL motifs of nuclear receptor coactivators. We demonstrated that in the context of the AR FXXLF motif many different amino acid residues at positions +2 and +3 are compatible with strong AR LBD interaction, although a preference for E at +2 and K or R at +3 was found. Pairwise systematic analysis of F/L swaps at +1 and +5 in FXXLF and LXXLL motifs showed: 1) F to L substitutions in natural FXXLF motifs abolished AR LBD interaction; 2) binding of interacting LXXLL motifs was unchanged or increased upon L to F substitutions; 3) certain noninteracting LXXLL motifs became strongly AR-interacting FXXLF motifs; whereas 4) other nonbinders remained unaffected by L to F substitutions. All FXXLF motifs, but not the corresponding LXXLL motifs, displayed a strong preference for AR LBD. Progesterone receptor LBD interacted with some FXXLF motifs, albeit always less efficiently than corresponding LXXLL motifs. AR LBD interaction of most FXXLF and LXXLL peptides depended on classical charge clamp residue K720, whereas E897 was less important. Other charged residues lining the AR coactivator-binding groove, K717 and R726, modulated optimal peptide binding. Interestingly, these four charged residues affected binding of individual peptides independent of an F or L at +1 and +5 in swap experiments. In conclusion, F residues determine strong and selective peptide interactions with AR. Sequences flanking the core motif determine the specific mode of FXXLF and LXXLL interactions.     

Three-dimensional structural model of the serine receptor ligand-binding domain.

Computer-based homology modeling techniques were used to construct a three-dimensional model of the Escherichia coli serine receptor ligand-binding domain based on the crystal structure of the Salmonella typhimurium aspartate receptor and the sequence homology between the two receptors. Residues that have been found in mutagenesis studies to be necessary for serine binding are located in a proposed serine-binding site. Several other mutations that affect swimming behavior require relatively small shifts in alpha-carbon positions in the model to give a minimized structure, suggesting that small changes in receptor conformation can affect the signaling state of the receptor.