Rolling adhesion of alphaL I domain mutants decorrelated from binding affinity

Activated lymphocyte function-associated antigen-1 (LFA-1, alphaLbeta2 integrin) found on leukocytes facilitates firm adhesion to endothelial cell layers by binding to intercellular adhesion molecule-1 (ICAM-1), which is up-regulated on endothelial cells at sites of inflammation. Recent work has shown that LFA-1 in a pre-activation, low-affinity state may also be involved in the initial tethering and rolling phase of the adhesion cascade. The inserted (I) domain of LFA-1 contains the ligand-binding epitope of the molecule, and a conformational change in this region during activation increases ligand affinity. We have displayed wild-type I domain on the surface of yeast and validated expression using I domain specific antibodies and flow cytometry. Surface display of I domain supports yeast rolling on ICAM-1-coated surfaces under shear flow. Expression of a locked open, high-affinity I domain mutant supports firm adhesion of yeast, while yeast displaying intermediate-affinity I domain mutants exhibit a range of rolling phenotypes. We find that rolling behavior for these mutants fails to correlate with ligand binding affinity. These results indicate that unstressed binding affinity is not the only molecular property that determines adhesive behavior under shear flow.     

Structure/function of the human glucocorticoid receptor: tyrosine 735 is important for transactivation.

Ligand-induced activation of the glucocorticoid receptor (GR) is not well understood. The GR ligand-binding domain was modeled, based on homology with the progesterone receptor. Tyrosine 735 interacts with the D ring of dexamethasone, and substitution of D ring functional groups results in partial agonist steroids with reduced ability to direct transactivation. Loss of the Tyr735 hydroxyl group by substitution to phenylalanine (Tyr735Phe) did not reduce ligand binding affinity [dissociation constant (Kd) 4.3 nM compared with Kd 4.6 nM for wild-type] and did not alter transrepression of an nuclear factor-kappaB (NF-kappaB reporter. But, there was a significant 30% reduction in maximal transactivation of a mouse mammary tumor virus (MMTV) reporter, although with an unchanged EC50 (8.6 nM compared with 6 nM). Substitution to a nonaromatic hydrophobic amino acid, valine (Tyr735Val), retained high-affinity ligand binding for dexamethasone (Kd 6 nM compared with 4.6 nM) and did not alter transrepression of NF-kappaB. However, there was a 36% reduction in MMTV activity with a right shift in EC50 (14.8 nM). The change to serine, a small polar amino acid (Tyr735Ser), caused significantly lower affinity for dexamethasone (10.4 nM). Maximal transrepression of NF-kappaB was unaltered, but the IC50 for this effect was increased. Tyr735Ser had a major shift in EC50 (118 nM) for transactivation of an MMTV reporter. Maximal transactivation of MMTV induced by the natural ligand cortisol was reduced to 60% by Tyr735Phe and Tyr735Val and was completely absent by Tyr735Ser. These data suggest that tyrosine 735 is important for ligand interpretation and transactivation.     

Expression, purification, and characterization of multiple, multifunctional human glucocorticoid receptor proteins

The glucocorticoid receptor (GR) is a nuclear receptor protein that plays a central role in glucose homeostasis, the stress response, control of the hypothalamic-pituitary-adrenal axis, and immuno-inflammatory processes via binding of the natural steroid, cortisol. GR is a well-validated drug target and continues to be an important target for new drug discovery efforts. Here, we describe a basic and simple method for Escherichia coli expression and purification of a variety of human GR proteins that contain all three of the functional domains of the protein: the activation function-1 domain, the DNA-binding domain, and the ligand-binding domain. We present characterization data to show that these purified, multifunctional GR proteins are active for ligand, coactivator, and DNA-binding. The work presented here should serve as a reference for future mechanistic, structural and drug discovery efforts that require purified, full or near full length, GR protein.     

Evidence for DNA-binding domain--ligand-binding domain communications in the androgen receptor

DNA binding as well as ligand binding by nuclear receptors has been studied extensively. Both binding functions are attributed to isolated domains of which the structure is known. The crystal structure of a complete receptor in complex with its ligand and DNA-response element, however, has been solved only for the peroxisome proliferator-activated receptor γ (PPARγ)-retinoid X receptor α (RXRα) heterodimer. This structure provided the first indication of direct interactions between the DNA-binding domain (DBD) and ligand-binding domain (LBD). In this study, we investigated whether there is a similar interface between the DNA- and ligand-binding domains for the androgen receptor (AR). Despite the structural differences between the AR- and PPARγ-LBD, a combination of in silico modeling and docking pointed out a putative interface between AR-DBD and AR-LBD. The surfaces were subjected to a point mutation analysis, which was inspired by known AR mutations described in androgen insensitivity syndromes and prostate cancer. Surprisingly, AR-LBD mutations D695N, R710A, F754S, and P766A induced a decrease in DNA binding but left ligand binding unaffected, while the DBD-residing mutations K590A, K592A, and E621A lowered the ligand-binding but not the DNA-binding affinity. We therefore propose that these residues are involved in allosteric communications between the AR-DBD and AR-LBD.     

Binding of brain-derived neurotrophic factor to the nerve growth factor receptor

The neurotrophic proteins BDNF and NGF are related in their primary structures, and both have high- and low-affinity receptors on their responsive neurons. In this study, we investigate the extent to which these receptors can discriminate between BDNF and NGF. We found that a 1000-fold excess of the heterologous ligand is needed to reduce binding to the high-affinity receptor by 50%, but that the same concentrations of BDNF and NGF similarly reduce the binding of either ligand to the low-affinity receptor. Results obtained with cells transfected with the low-affinity NGF receptor gene indicate that these cells bind BDNF, in addition to NGF, whereas cells before transfection do not. These data indicate that the low-affinity NGF receptor is also a low-affinity BDNF receptor and that whatever is conferring high-affinity binding and biological response also considerably reinforces the ability of the low-affinity receptor to discriminate between NGF and BDNF.     

A chimeric Cre recombinase inducible by synthetic,but not by natural ligands of the glucocorticoid receptor.

We have developed a new ligand-dependent chimeric recombinase (Cre-GRdex) by fusing the site-specific Cre recombinase to the ligand binding domain (LBD) of a mutant human glucocorticoid receptor (GRdex). The synthetic glucocorticoid receptor (GR) ligands dexamethasone, triamcinolone acetonide and RU38486efficiently induce recombinase activity in F9 murine embryonal carcinoma cells expressing constitutively Cre-GRdex. In contrast, no recombinase activity was detected in the absence of ligand or in the presence of the natural GR ligands corticosterone, cortisol or aldosterone. Moreover, physiological concentrations of these natural GR ligands do not affect Cre-GRdexrecombinase activity induced by dexamethasone. Thus, as previously shown using Cre-oestrogen receptor (ER) fusion proteins, Cre-GRdexmight be useful for achieving loxP site-directed mutagenesis in cultured cells and spatio-temporally controlled somatic cell mutagenesis in transgenic mice.     

Steroid induction of low-affinity glucocorticoid binding sites in rat liver microsomes

Rat liver contains two glucocorticoid binding sites: the high-affinity or glucocorticoid receptor (GR) and the low-affinity glucocorticoid binding sites, or LAGS. The Kd of LAGS predicts that they can be half-saturated by plasma corticosteroids in some physiological circumstances and, therefore, that they can play relevant roles in the rat liver. [3H]dexamethasone was used as a ligand in exchange assays, to study the relative abundance of GR and LAGS in cell fractions of rat liver. GR were found in the cytosol, but not in the purified nuclei, the mitochondria, or the microsomes. LAGS were found in all the particulate fractions, being more abundant in the smooth-surfaced microsomes, but they were not found in the cytosol. The LAGS of microsomes and purified nuclei showed the same Kd and also the same broad range of steroid competition with [3H]dexamethasone (cortisol = progesterone greater than dexamethasone greater than or equal to corticosterone greater than R5020 greater than DHEA greater than testosterone = estradiol). LAGS were found in liver, placenta and kidney, but not in other GR-containing organs. This suggests that the LAGS could be involved in physiological functions related to the metabolism of steroid hormones. The liver microsome LAGS were undetectable at rat birth, and became present in the 25-day-old rat. The level of LAGS then increased progressively, reaching its maximum level in the 2-3-month-old rats (10 pmol/mg protein), and declining afterwards to reach the adulthood level (5 pmol/mg protein) in 6-month-old rats. LAGS are mainly controlled by the corticoadrenal steroids, which is shown by their dramatic decrease after adrenalectomy, and especially after hypophysectomy. Many steroid hormones, like estradiol, testosterone, and corticosterone (but not progesterone) induce LAGS, estradiol being the most effective. A combination of T4 and corticosterone was more effective in inducing LAGS than when the two hormones were injected separately. It is possible to conclude that rat liver LAGS are mainly microsomal proteins, whose concentration is regulated by a multihormone system under pituitary control.     

Conformational dynamics and molecular recognition: backbone dynamics of the estrogen receptor DNA-binding domain.

We examined the internal mobility of the estrogen receptor DNA-binding domain (ER DBD) using NMR15N relaxation measurements and compared it to that of the glucocorticoid receptor DNA-binding domain (GR DBD). The studied protein fragments consist of residues Arg183-His267 of the human ER and residues Lys438-Gln520 of the rat GR. The15N longitudinal (R1) and transverse (R2) relaxation rates and steady state {1H}-15N nuclear Overhauser enhancements (NOEs) were measured at 30 degrees C at1H NMR frequencies of 500 and 600 MHz. The NOE versus sequence profile and calculated order parameters for ER DBD backbone motions indicate enhanced internal dynamics on pico- to nanosecond time-scales in two regions of the core DBD. These are the extended strand which links the DNA recognition helix to the second zinc domain and the larger loop region of the second zinc domain. The mobility of the corresponding regions of the GR DBD, in particular that of the second zinc domain, is more limited. In addition, we find large differences between the ER and GR DBDs in the extent of conformational exchange mobility on micro- to millisecond time-scales. Based on measurements of R2as a function of the15N refocusing (CPMG) delay and quantitative (Lipari-Szabo-type) analysis, we conclude that conformational exchange occurs in the loop of the first zinc domain and throughout most of the second zinc domain of the ER DBD. The conformational exchange dynamics in GR DBD is less extensive and localized to two sites in the second zinc domain. The different dynamical features seen in the two proteins is consistent with previous studies of the free state structures in which the second zinc domain in the ER DBD was concluded to be disordered whereas the corresponding region of the GR DBD adopts a stable fold. Moreover, the regions of the ER DBD that undergo conformational dynamics on the micro- to millisecond time-scales in the free state are involved in intermolecular protein-DNA and protein-protein interactions in the dimeric bound state. Based on the present data and the previously published dynamical and DNA binding properties of a GR DBD triple mutant which recognize an ER binding site on DNA, we argue that the free state dynamical properties of the nuclear receptor DBDs is an important element in molecular recognition upon DNA binding.     

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.     

A Charge-inverting Mutation in the “Linker” Region of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Alters Agonist Binding and Gating Kinetics Independently of Allosteric Modulators

AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728–4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators.     

A two-module region of the low-density lipoprotein receptor sufficient for formation of complexes with apolipoprotein E ligands

The low-density lipoprotein (LDL) receptor transports two different classes of cholesterol-carrying lipoprotein particles into cells: LDL particles, which contain a single copy of apolipoprotein B-100 (apoB-100), and beta-migrating very low-density lipoprotein (beta-VLDL) particles, which contain multiple copies of apolipoprotein E (apoE). The ligand-binding domain of the receptor lies at its amino-terminal end within seven adjacent LDL-A repeats (LA1-LA7). Although prior work clearly establishes that LA5 is required for high-affinity binding of particles containing apolipoprotein E (apoE), the number of ligand-binding repeats sufficient to bind apoE ligands has not yet been determined. Similarly, uncertainty exists as to whether a single lipid-activated apoE receptor-binding site within a particle is capable of binding to the LDLR with high affinity or whether more than one is required. Here, we establish that the LA4-5 two-repeat pair is sufficient to bind apoE-containing ligands, on the basis of binding studies performed with a series of LDLR-derived "minireceptors" containing up to four repeats. Using single chain multimers of the apoE receptor-binding domain (N-apoE), we also show that more than one receptor-binding site in its lipid-activated conformation is required to bind to the LDLR with high affinity. Thus, in addition to inducing a conformational change in the structure of N-apoE, lipid association enhances the affinity of apoE for the LDLR in part by creating a multivalent ligand.     

Determinants of high-affinity DNA binding by the glucocorticoid receptor: evaluation of receptor domains outside the DNA-binding domain.

In this study, we have investigated the influence of regions outside the DNA-binding domain of the human glucocorticoid receptor on high-affinity DNA binding. We find that the DNA-binding domain shows a 10-fold lower affinity for a palindromic DNA-binding site than the intact receptor. The N-terminal part of the receptor protein does not influence its DNA-binding affinity, while the C-terminal steroid-binding domain increases the DNA-binding affinity of the receptor molecule. It has previously been shown that both the intact glucocorticoid receptor and the glucocorticoid receptor DNA-binding domain bind to a palindromic glucocorticoid response element on DNA as dimers. It is likely that differences in DNA-binding affinity observed result from protein-protein interactions outside the DNA-binding domain between receptor monomers, as has been shown for the estrogen receptor. We have previously identified a segment involved in protein-protein interactions between DNA-binding domains of glucocorticoid receptors. This, in combination with results presented in this study, suggests that there are at least two sites of contact between receptor monomers bound to DNA. We suggest that the interaction between the DNA-binding domains may act primarily to restrict DNA binding to binding sites with appropriate half-site spacing and that additional stability of the receptor dimer is provided by the interactions between the steroid-binding domains.