Homology-modeled ligand-binding domains of medaka estrogen receptors and androgen receptors: A model system for the study of reproduction

Estrogen and androgen and their receptors play critical roles in physiological processes such as sexual differentiation and development. Using the available structural models for the human estrogen receptors alpha and beta and androgen receptor as templates, we designed in silico agonist and antagonist models of medaka estrogen receptor (meER) alpha, beta-1, and beta-2, and androgen receptor (meAR) alpha and beta. Using these models, we studied (1) the structural relationship between the ligand-binding domains (LBDs) of ERs and ARs of human and medaka, and (2) whether medaka ER and AR can be potential models for studying the ligand-binding activities of various agonists and antagonists of these receptors by docking analysis. A high level of conservation was observed between the sequences of the ligand-binding domains of meERα and huERα, meERβ1 and huERβ, meERβ2, and huERβ with 62.8%, 66.4%, and 65.1% identity, respectively. The sequence conservation between meARα and huAR, meARβ, and huAR was found with 70.1% and 61.0% of identity, respectively. Thirty-three selected endocrine disrupting chemicals (EDCs), including both agonists and antagonists, were docked into the LBD of ER and AR, and the corresponding docking score for medaka models and human templates were calculated. In order to confirm the conservation of the overall geometry and the binding pocket, the backbone root mean square deviation (RMSD) for Cα atoms was derived from the structure superposition of all 10 medaka homology models to the six human templates. Our results suggested conformational conservation between the ERs and ARs of medaka and human, Thus, medaka could be highly useful as a model system for studies involving estrogen and androgen interaction with their receptors.     

A point mutation in the seventh hydrophobic domain of the alpha 2 adrenergic receptor increases its affinity for a family of beta receptor antagonists.

Previous studies have shown that differences in subtype-specific ligand binding between alpha 2 and beta 2 adrenergic receptors are largely determined by the seventh hydrophobic domain. Here, we report that a single amino acid substitution (Phe412----Asn) in the seventh hydrophobic domain of the alpha 2 adrenergic receptor reduces affinity for the alpha 2 antagonist yohimbine by 350-fold and increases affinity for beta antagonist alprenolol by 3000-fold. The affinity of this mutant receptor alpha 2F----N for several alpha and beta adrenergic receptor agonists and antagonists was determined. Beta adrenergic receptor antagonists containing an oxygen atom linking the amino side chain with the aromatic ring bound to alpha 2F----N with high affinity, while the beta receptor antagonist sotalol, which lacks this oxygen, bound with low affinity. These data suggest that the Asn residue is involved in conferring specificity for binding to a specific class of beta receptor antagonists.     

Pancreatic insulin content regulation by the estrogen receptor ER alpha

The function of pancreatic beta-cells is the synthesis and release of insulin, the main hormone involved in blood glucose homeostasis. Estrogen receptors, ER alpha and ER beta, are important molecules involved in glucose metabolism, yet their role in pancreatic beta-cell physiology is still greatly unknown. In this report we show that both ER alpha and ER beta are present in pancreatic beta-cells. Long term exposure to physiological concentrations of 17beta-estradiol (E2) increased beta-cell insulin content, insulin gene expression and insulin release, yet pancreatic beta-cell mass was unaltered. The up-regulation of pancreatic beta-cell insulin content was imitated by environmentally relevant doses of the widespread endocrine disruptor Bisphenol-A (BPA). The use of ER alpha and ER beta agonists as well as ER alphaKO and ER betaKO mice suggests that the estrogen receptor involved is ER alpha. The up-regulation of pancreatic insulin content by ER alpha activation involves ERK1/2. These data may be important to explain the actions of E2 and environmental estrogens in endocrine pancreatic function and blood glucose homeostasis.     

Multiple classes of dopamine receptors in mammalian central nervous system: the involvement of dopamine-sensitive adenylyl cyclase.

Two classes of dopamine receptor mechanism are defined according to their association with, or independence from, a dopamine-sensitive adenylyl cyclase. Dopamine receptors unrelated to adenylyl cyclase are designated type alpha. Dopamine receptors linked to adenylyl cyclase are designated type beta. Drugs discriminate between the two receptor mechanisms. The dopaminergic ergots (lisuride, lergotrile and CB-154) and their antagonists (such as metoclopramide) are relatively specific for the alpha-dopaminergic receptor in the anterior pituitary. Other agonists (e.g. apomorphine and dopamine) and antagonists (e.g. antipsychotic phenothiazines and butyrophenones) affect both classes of receptor.     

Activity of cytisine and its brominated isosteres on recombinant human alpha7, alpha4beta2 and alpha4beta4 nicotinic acetylcholine receptors

Effects of cytisine (cy), 3-bromocytisine (3-Br-cy), 5-bromocytisine (5-Br-cy) and 3,5-dibromocytisine (3,5-diBr-cy) on human (h) alpha7-, alpha4beta2- and alpha4beta4 nicotinic acetylcholine (nACh) receptors, expressed in Xenopus oocytes and cell lines, have been investigated. Cy and its bromo-isosteres fully inhibited binding of both [alpha-(125)I]bungarotoxin ([alpha-(125)I]BgTx) to halpha7- and [(3)H]cy to halpha4beta2- or halpha4beta4-nACh receptors. 3-Br-cy was the most potent inhibitor of both [alpha-(125)I]BgTx and [(3)H]cy binding. Cy was less potent than 3-Br-cy, but 5-Br-cy and 3,5-diBr-cy were the least potent inhibitors. Cy and 3-Br-cy were potent full agonists at halpha7-nACh receptors but behaved as partial agonists at halpha4beta2- and halpha4beta4-nACh receptors. 5-Br-cy and 3,5-diBr-cy had low potency and were partial agonists at halpha7- and halpha4beta4-nACh receptors, but they elicited no responses on halpha4beta2-nACh receptors. Cy and 3-Br-cy produced dual dose-response curves (DRC) at both halpha4beta2- and halpha4beta4-nACh receptors, but ACh produced dual DRC only at halpha4beta2-nACh receptors. Low concentrations of cy, 3-Br-cy and 5-Br-cy enhanced ACh responses of oocytes expressing halpha4beta2-nACh receptors, but at high concentrations they inhibited the responses. In contrast, 3,5-diBr-cy only inhibited, in a competitive manner, ACh responses of halpha4beta2-nACh receptors. It is concluded that bromination of the pyridone ring of cy produces marked changes in effects of cy that are manifest as nACh receptor subtype-specific differences in binding affinities and in functional potencies and efficacies.     

Ligands "activate" integrin alpha IIb beta 3 (platelet GPIIb-IIIa)

Integrin alpha IIb beta 3 (platelet GPIIb-IIIa) binds fibrinogen via recognition sequences such as Arg-Gly-Asp (RGD). Fibrinogen binding requires agonist activation of platelets, whereas the binding of short synthetic RGD peptides does not. We now find that RGD peptide binding leads to changes in alpha IIb beta 3 that are associated with acquisition of high affinity fibrinogen-binding function (activation) and subsequent platelet aggregation. The structural specificities for peptide activation and for inhibition of ligand binding are similar, indicating that both are consequences of occupancy of the same site(s) on alpha IIb beta 3. Thus, the RGD sequence is a trigger of high affinity ligand binding to alpha IIb beta 3, and certain RGD-mimetics are partial agonists as well as competitive antagonists of integrin function.     

Regulation of alpha- and beta-adrenergic receptor subclasses by gonadal steroids in human myometrium

Both alpha- and beta-adrenergic receptors have been identified in the human myometrium by radioligand binding. Both adrenergic receptor subclasses have been shown to mediate the contractile response of the uterus upon catecholamine stimulation: alpha-adrenergic receptors cause uterine contraction while beta-adrenergic receptors induce relaxation. We have identified alpha 1- and alpha 2-adrenergic receptors in myometrial membranes using the newly developed radiolabelled specific antagonists [3H]-prazosin and [3H]-rauwolscine. This enabled us to characterize both receptor subclasses individually. Beta adrenergic receptors were identified using the radiolabelled antagonist (-)-[3H]-dihydroalprenolol. Binding of radioligands to the myometrial membrane receptors was rapid, readily reversible, of high affinity and stereoselective. The total number of alpha 1-, alpha 2- and beta-receptors was determined by Scatchard analysis of radioligand saturation binding and the beta/beta 2-receptor ratio was determined by computer analysis of the beta 2-selective antagonist ICI 118 551) (-)-[3H]-dihydroalprenolol competition binding curves. This enabled us to study the regulation of both alpha- and beta-receptor subclasses under various physiological and pharmacological conditions in the human, i.e., during different phases of the menstrual cycle, in postmenopausal women and during depo-progestin (Medroxyprogesterone acetate) therapy. Only the alpha 2- and beta 1-adrenergic receptor concentrations were found to be subjected to gonadal steroid regulation. The number of alpha 2- and beta 1-adrenergic receptors increased concomitantly with circulating plasma oestradiol levels. This effect was counteracted by progesterone. The number of alpha 1- and beta 2-adrenergic receptors was unaffected by the gonadal steroid environment. These results are an example of the heteroregulation of membrane receptors by oestrogens and progesterone and cast new light on the regulatory mechanisms involved in uterine contractility in the human.     

Activity of alpha7-selective agonists at nicotinic and serotonin 5HT3 receptors expressed in Xenopus oocytes

Nicotinic receptors containing alpha7 subunits are widely distributed in the central nervous system and are thought to be involved in a number of functions. However, it has been difficult to study alpha7-containing receptors in vivo because of a paucity of selective agonists. A new spirooxazolidinone compound, AR-R17779, was recently described as potent agonist at alpha7 receptors, but electrophysiological studies at other types of nicotinic receptors have not been carried out. We characterized the activity of AR-R17779 at alpha7, alpha4beta2, alpha3beta4, alpha3beta2, alpha3beta2alpha5 receptors expressed in Xenopus oocytes. In addition, since there is significant homology between nicotinic alpha7 and serotonin 5HT(3) receptors, the activity of AR-R17779 at expressed 5HT(3a) receptors was also examined. Finally, actions of tropisetron and ondansetron, two 5HT(3) antagonists, were explored. AR-R17779 was found to activate alpha7 receptors, but had no activity at other types of nicotinic receptors, and also had no activity at 5HT(3a) receptors. Tropisetron activated, while ondansetron acted as an antagonist, at alpha7 nicotinic receptors. The two 5HT(3) antagonists also acted as antagonists at alpha4beta2 and alpha3beta4 nicotinic receptors. Thus, AR-R17779 was confirmed to be a selective nicotinic alpha7 receptor agonist and to be without activity at 5HT(3) receptors. In contrast, the actions of tropisetron and ondansetron on nicotinic receptors were complex.     

(+/-)-[3H]Epinephrine and (-)[3H]dihydroalprenolol binding to beta1- and beta2-noradrenergic receptors in brain, heart, and lung membranes.

(+/-)-[3H]Epinephrine binds to beta-receptors in calf cerebellar and rat lung membranes in the presence of 1.0 mM pyrocatechol and 1.0 microM phentolamine, with dissociation constants at 4 degrees C of 11 nM and 24 nM, respectively. (+/-)-[3H]Epinephrine associates to equilibrium within 20 min in both tissues, and over 50% of the binding is rapidly dissociable. Inhibition of binding by agonists and antagonists is highly stereoselective, and the structure-activity relationships of adrenergic agents in inhibiting (+/-)-[3H]epinephrine binding suggest an interaction with beta2 type noradrenergic receptors. (-)-Isoproterenol has an apparent Ki of 2 nM, (-)-epinephrine is 1.5 to 3 times weaker, and (-)-norepinephrine is 30 to 60 times weaker. Salbutamol and terbutaline, selective beta2-agonists, are potent inhibitors of binding, as are several nonspecific antagonists. Properties of the sites labeled by (+/-)-[3H]epinephrine in calf cerebellum and rat lung are closely similar. (-)-[3H]Dihydroalprenolol binding in calf cerebellum and rat lung also shows beta2 characteristics. Antagonists have similar potencies in inhibiting (-)-[3H]dihydroalprenolol and (+/-)-[3H]epinephrine binding in both tissues, but agonists are in general more potent inhibitors of (+/-)-[3H]epinephrine. Sodium and lithium selectively lower the affinity of (+/-)-[3H]epinephrine at its binding sites and the affinities of agonists, but not antagonists, at the (-)-[3H]dihydroalprenolol site. Specific (+/-)-[3H]epinephrine binding was not detectable in calf cortex and rat heart, where (-)-[3H]dihydroalprenolol binding suggests a beta1-receptor. A physiological significance of (+/-)-[3H]epinephrine binding is suggested by the strong correlation for agonists and antagonists between affinities in inhibiting binding, and in stimulating or inhibiting a beta-receptor-coupled adenylate cyclase in frog erythrocytes.     

beta-Adrenergic regulation of insulin and epidermal growth factor receptors in rat adipocytes

Incubation of intact rat adipocytes with physiological concentrations of catecholamines inhibits the specific binding of 125I-insulin and 125I-epidermal growth factor (EGF) by 40 to 70%. Affinity labeling of the alpha subunit of the insulin receptor demonstrates that the inhibition of hormone binding is directly reflective of a specific decrease in the degree of receptor occupancy. The stereospecificity and dose dependency of the binding inhibitions are typical of a classic beta 1-adrenergic receptor response with half-maximal inhibition occurring at 10 nM R-(-)-isoproterenol. Specific alpha-adrenergic receptor agonists and beta-adrenergic receptor antagonists have no effect, while beta-adrenergic receptor antagonists block the inhibition of 125I-insulin and 125I-EGF binding to receptors induced by beta-adrenergic receptor agonists. Further, these effects are mimicked by incubation of adipocytes with dibutyryl cyclic AMP or with 3-isobutyl-1-methylxanthine. The beta-adrenergic inhibition of both 125I-insulin and 125I-EGF binding is very rapid, requiring only 10 min of isoproterenol pretreatment at 37 degrees C for a maximal effect. Removal of isoproterenol by washing the cells in the presence of alprenolol leads to complete reversal of these effects. The inhibition of 125I-EGF binding is temperature dependent whereas the inhibition of 125I-insulin binding is relatively insensitive to the temperature of isoproterenol pretreatment. Scatchard analysis of 125I-insulin and 125I-EGF binding demonstrated that the decrease of insulin receptor-binding activity may be due to a decrease in the apparent number of insulin receptors while the inhibition of EGF receptor binding can be accounted for by a decrease in apparent EGF receptor affinity. The decrease in the insulin receptor-binding activity is physiologically expressed as a dose-dependent decrease of insulin responsiveness in the adipocyte with respect to two known responses, stimulation of insulin-like growth factor II receptor binding and activation of the glucose-transport system. These results demonstrate a beta-adrenergic receptor-mediated cyclic AMP-dependent mechanism for the regulation of insulin and EGF receptors in the rat adipocyte.     

Preponderance of alpha 2- over beta 1-adrenergic receptor sites in human fat cells is not predictive of the lipolytic effect of physiological catecholamines

Adrenergic control of human fat cell lipolysis is mediated by two kinds of receptor sites that are simultaneously stimulated by physiological amines. To establish a correlation between the binding characteristics of the receptor and biological functions, the ability of physiological amines to stimulate or inhibit isolated fat cell lipolysis in vitro was compared to the beta- and alpha 2-adrenoceptor properties of the same fat cell batch. The beta-selective antagonist (-)[3H]dihydroalprenolol ([3H]DHA) and the alpha 2-selective antagonists [3H]yohimbine ([3H]YOH) and [3H]rauwolscine ([3H]RAU) were used to identify and characterize the two receptor sites. Binding of each ligand was rapid, saturable, and specific. The results demonstrate 1) the weaker lipolytic effect of epinephrine compared with norepinephrine. This can be explained by the equipotency of the amines at the beta 1-sites and the higher affinity of epinephrine for alpha 2-adrenergic receptors. 2) The preponderance of alpha 2-adrenergic receptor sites labeled by [3H]YOH (Bmax, 586 +/- 95 fmol/mg protein; KD, 2.7 +/- 0.2 nM) or [3H]RAU (Bmax, 580 +/- 100 fmol/mg protein; KD, 3.7 +/- 0.1 nM). These two ligands can be successfully used to label alpha 2-adrenergic receptor sites. 3) The beta 1-adrenergic receptor population labeled by [3H]DHA(Bmax, 234 +/- 37 fmol/mg protein; KD, 1.8 +/- 0.4 nM), although a third as numerous as the alpha 2-adrenergic population, is responsible for the lipolytic effect of physiological amines and is weakly counteracted by simultaneous alpha 2-adrenergic receptor stimulation under our experimental conditions. It is concluded that, in human fat cells, the characterization of beta 1- and alpha 2-adrenergic receptors by saturation studies or kinetic analysis to determine affinity (KD) and maximal number of binding sites (Bmax) is not sufficient for an accurate characterization of the functional adrenergic receptors involved in the observed biological effect.     

alpha4beta3delta GABA(A) receptors characterized by fluorescence resonance energy transfer-derived measurements of membrane potential

Selective modulators of gamma-aminobutyric acid, type A (GABA(A)) receptors containing alpha(4) subunits may provide new treatments for epilepsy and premenstrual syndrome. Using mouse L(-tk) cells, we stably expressed the native GABA(A) receptor subunit combinations alpha(3)beta(3)gamma(2,) alpha(4)beta(3)gamma(2), and, for the first time, alpha(4)beta(3)delta and characterized their properties using a novel fluorescence resonance energy transfer assay of GABA-evoked depolarizations. GABA evoked concentration-dependent decreases in fluorescence resonance energy transfer that were blocked by GABA(A) receptor antagonists and, for alpha(3)beta(3)gamma(2) and alpha(4)beta(3)gamma(2) receptors, modulated by benzodiazepines with the expected subtype specificity. When combined with alpha(4) and beta(3), delta subunits, compared with gamma(2), conferred greater sensitivity to the agonists GABA, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), and muscimol and greater maximal efficacy to THIP. alpha(4)beta(3)delta responses were markedly modulated by steroids and anesthetics. Alphaxalone, pentobarbital, and pregnanolone were all 3-7-fold more efficacious at alpha(4)beta(3)delta compared with alpha(4)beta(3)gamma(2.) The fluorescence technique used in this study has proven valuable for extensive characterization of a novel GABA(A) receptor. For GABA(A) receptors containing alpha(4) subunits, our experiments reveal that inclusion of delta instead of gamma(2) subunits can increase the affinity and in some cases the efficacy of agonists and can increase the efficacy of allosteric modulators. Pregnanolone was a particularly efficacious modulator of alpha(4)beta(3)delta receptors, consistent with a central role for this subunit combination in premenstrual syndrome.     

Association with BiP and aggregation of class II MHC molecules synthesized in the absence of invariant chain.

Class II molecules of the major histocompatibility complex (MHC) are composed of two polymorphic glycoprotein chains (alpha and beta), that associate in the ER with a third, non-polymorphic glycoprotein known as the invariant chain (Ii). We have examined the relationship between the intracellular transport and physico-chemical characteristics of various combinations of murine alpha, beta and Ii chains. Biochemical and morphological analyses of transfected fibroblasts expressing class II MHC chains show that both unassembled alpha and beta chains, as well as a large fraction of alpha+beta complexes synthesized in the absence of Ii chain, are retained in the ER in association with the immunoglobulin heavy chain binding protein, BiP. Analyses by sedimentation velocity on sucrose gradients show that most incompletely assembled class II MHC species exist as high molecular weight aggregates in both transfected fibroblasts and spleen cells from mice carrying a disruption of the Ii chain gene. This is in contrast to the sedimentation properties of alpha beta Ii complexes from normal mice, which migrate as discrete, stoichiometric complexes of M(r) approximately 200,000-300,000. These observations suggest that assembly with the Ii chain prevents accumulation of aggregated alpha and beta chains in the ER, which might relate to the known ability of the Ii chain to promote exit of class II MHC molecules from the ER.     

A small molecule agonist of an integrin, alphaLbeta2

The binding of integrin alpha(L)beta(2) to its ligand intercellular adhesion molecule-1 is required for immune responses and leukocyte trafficking. Small molecule antagonists of alpha(L)beta(2) are under intense investigation as potential anti-inflammatory drugs. We describe for the first time a small molecule integrin agonist. A previously described alpha/beta I allosteric inhibitor, compound 4, functions as an agonist of alpha(L)beta(2) in Ca(2+) and Mg(2+)and as an antagonist in Mn(2+). We have characterized the mechanism of activation and its competitive and noncompetitive inhibition by different compounds. Although it stimulates ligand binding, compound 4 nonetheless inhibits lymphocyte transendothelial migration. Agonism by compound 4 results in accumulation of alpha(L)beta(2) in the uropod, extreme uropod elongation, and defective de-adhesion. Small molecule integrin agonists open up novel therapeutic possibilities.     

A new fluorescence complementation biosensor for detection of estrogenic compounds

Estrogenic compounds are an important class of hormonal substances that can be found as environmental contaminants, with sources including pharmaceuticals, human and animal waste, the chemical industry, and microbial metabolism. Here we report the creation of a biosensor useful for monitoring such compounds, based on complementation of fluorescent protein fragments. A series of sensors were made consisting of fragments of a split mVenus fluorescent protein fused at several different N-terminal and C-terminal positions flanking the ligand binding domain of the estrogen receptor alpha. When expressed in HeLa cells, sensor 6 (ERα 312-595) showed a nine-fold increase in fluorescence in the presence of estrogen receptor agonists or antagonists. Sensor 2 (ERα 281-549) discriminated between agonists and antagonists by showing a decrease in fluorescence in the presence of agonists while being induced by antagonists. The fluorescent signal of sensor 6 increased over a period of 24 h, with a two-fold induction visible at 4 h and four-fold at 8 h of ligand incubation. Ligand titration showed a good correlation with the known relative binding affinities of the compound. The sensor could detect a number of compounds of interest that can act as environmental endocrine disruptors. The lack of a substrate requirement, the speed of signal development, the potential for high throughput assays, and the ability to distinguish agonists from antagonists make this an attractive sensor for widespread use.     

ERβ Binds N-CoR in the Presence of Estrogens via an LXXLL-like Motif in the N-CoR C-terminus

Nuclear receptors (NRs) usually bind the corepressors N-CoR and SMRT in the absence of ligand or in the presence of antagonists. Agonist binding leads to corepressor release and recruitment of coactivators. Here, we report that estrogen receptor beta (ERbeta) binds N-CoR and SMRT in the presence of agonists, but not antagonists, in vitro and in vivo. This ligand preference differs from that of ERalpha interactions with corepressors, which are inhibited by estradiol, and resembles that of ERbeta interactions with coactivators. ERbeta /N-CoR interactions involve ERbeta AF-2, which also mediates coactivator recognition. Moreover, ERbeta recognizes a sequence (PLTIRML) in the N-CoR C-terminus that resembles coactivator LXXLL motifs. Inhibition of histone deacetylase activity specifically potentiates ERbeta LBD activity, suggesting that corepressors restrict the activity of AF-2. We conclude that the ER isoforms show completely distinct modes of interaction with a physiologically important corepressor and discuss our results in terms of ER isoform specificity in vivo.