A novel progestogen receptor subtype in the Japanese eel, Anguilla japonica.

A cDNA encoding a second type of a progestogen receptor (ePR2) was isolated from the same library as we had previously cloned a functional PR (ePR1) in eel testis. The amino acid sequence of the ePR2 shows low homology with ePR1 (34%), but both PRs showed progestogen-dependent transactivation in transfection experiment. Tissue distribution of ePR2 mRNA was clearly different from that of ePR1. Protein interaction between two PRs was demonstrated in vitro by a glutathione S-transferase pull-down assay. These results indicate that ePR2 is also a functional PR. This is the first isolation of two different functional PR molecules from a vertebrate.     

Heterodimerization of alpha 2A- and beta 1-adrenergic receptors

beta- and alpha(2)-adrenergic receptors are known to exhibit substantial cross-talk and mutual regulation in tissues where they are expressed together. We have found that the beta(1)-adrenergic receptor (beta(1)AR) and alpha(2A)-adrenergic receptor (alpha(2A)AR) heterodimerize when coexpressed in cells. Immunoprecipitation studies with differentially tagged beta(1)AR and alpha(2A)AR expressed in HEK-293 cells revealed robust co-immunoprecipitation of the two receptors. Moreover, agonist stimulation of alpha(2A)AR was found to induce substantial internalization of coexpressed beta(1)AR, providing further evidence for a physical association between the two receptors in a cellular environment. Ligand binding assays examining displacement of [(3)H]dihydroalprenolol binding to the beta(1)AR by various ligands revealed that beta(1)AR pharmacological properties were significantly altered when the receptor was coexpressed with alpha(2A)AR. Finally, beta(1)AR/alpha(2A)AR heterodimerization was found to be markedly enhanced by a beta(1)AR point mutation (N15A) that blocks N-linked glycosylation of the beta(1)AR as well as by point mutations (N10A/N14A) that block N-linked glycosylation of the alpha(2A)AR. These data reveal an interaction between beta(1)AR and alpha(2A)AR that is regulated by glycosylation and that may play a key role in cross-talk and mutual regulation between these receptors.     

Receptor binding and transactivation activities of red clover isoflavones and their metabolites

Red clover extracts contain a variety of isoflavones, which have affinity toward estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta), androgen receptor (AR), and progesterone receptor (PR). Upon ingestion, they undergo various metabolic transformations. For a complete evaluation of red clover extracts and possible health benefits, the resulting metabolites should also be investigated. Biochanin A, formononetin, genistein, daidzein, dihydrobiochanin A, dihydroformononetin, dihydrogenistein, dihydrodaidzein, 3'-hydroxygenistein, 6-hydroxydaidzein, 6-hydroxydesmethylangolensin, equol, O-desmethylangolensin, angolensin, and p-ethylphenol were tested for their transactivation potential toward ERalpha, AR, and PR in yeast. Competitive binding assays with radiolabeled 17beta-estradiol, 17alpha-methyltrienolone or progesterone assessed binding to the respective ERalpha and ERbeta, AR, and PR. The compounds showed only weak binding affinity to AR and PR, with IC(50) values being greater (i.e., lesser affinity) than 10(-5)M for the respective receptor. So far, beneficial health effects have been attributed to the production of equol. We propose that other metabolites can also contribute to these effects. However, more detailed information for the formation of these metabolites in humans and for bioavailability data are required to confirm our assumptions.     

Resistance of the human beta 1-adrenergic receptor to agonist-mediated down-regulation. Role of the C terminus in determining beta-subtype degradation

Prolonged agonist stimulation results in down-regulation of most G protein-coupled receptors. When we exposed baby hamster kidney cells stably expressing the human beta1-adrenergic receptor (beta 1AR) to agonist over a 24-h period, we instead observed an increase of approximately 30% in both beta 1AR binding activity and immune-detected receptors. In contrast, beta 2AR expressed in these cells exhibited a decrease of > or =50%. We determined that the basal turnover rates of the two subtypes were similar (t(1/2) approximately 7 h) and that agonist stimulation increased beta 2AR but not beta 1AR turnover. Blocking receptor trafficking to lysosomes with bafilomycin A1 had no effect on basal turnover of either subtype but blocked agonist-stimulated beta 2AR turnover. As beta 1AR mRNA levels increased in agonist-stimulated cells, beta 1AR up-regulation appeared to result from increased synthesis with no change in degradation. To explore the basis for the subtype differences, we expressed chimeras in which the C termini had been exchanged. Each chimera responded to persistent agonist stimulation based on the source of its C-tail; beta 1AR with a beta 2AR C-tail underwent down-regulation, and beta 2AR with a beta 1AR C-tail underwent up-regulation. The C-tails had a corresponding effect on agonist-stimulated receptor phosphorylation and internalization with the order being beta 2AR > beta 1AR with beta 2AR C-tail > beta 2AR with a beta 1AR C-tail > beta 1AR. As internalization may be a prerequisite for down-regulation, we addressed this possibility by co-expressing each subtype with arrestin-2. Although beta 1AR internalization was increased to that of beta 2AR, down-regulation still did not occur. Instead, beta 1AR accumulated inside the cells. We conclude that in unstimulated cells, both subtypes appear to be turned over by the same mechanism. Upon agonist stimulation, both subtypes are internalized, and beta 2AR but not beta 1AR undergoes lysosomal degradation, the fate of each subtype being regulated by determinants in its C-tail.     

Genetic analysis of the molecular basis for beta-adrenergic receptor subtype specificity

Pharmacological analysis of ligand binding to the beta-adrenergic receptor (beta AR) has revealed the existence of two distinct receptor subtypes (beta 1 and beta 2) which are the products of different genes. The predicted amino acid sequences of the beta 1 and beta 2 receptors differ by 48%. To identify the regions of the proteins responsible for determining receptor subtype, chimeras were constructed from domains of the human beta 1 and hamster beta 2 receptors. Analysis of the ligand-binding characteristics of these hybrid receptors revealed that residues in the middle portion of the beta AR sequence, particularly around transmembrane regions 4 and 5, contribute to the subtype specific binding of agonists. Smaller molecular replacements of regions of the hamster beta 2 AR with the analogous regions from the avian beta 1 AR, however, failed to identify any single residue substitution capable of altering the subtype specificity of the receptor. These data indicate that, whereas sequences around transmembrane regions 4 and 5 may contribute to conformations which influence the ligand-binding properties of the receptor, the subtype-specific differences in amine-substituted agonist binding cannot be attributed to a single molecular interaction between the ligand and any amino acid residue which is divergent between the beta 1 and beta 2 receptors.     

Chromatography studies on bio-affinity of nine ligands of α1-adrenoceptor to α1D subtypes overexpressed in cell membrane

To improve selectivity and specificity of cell membrane chromatography (CMC), the chromatography affinities of nine ligands of α₁-adrenergic receptor(AR)to α_1D-AR subtype were investigated. The human embryonic kidney (HEK) 293 cells expressed by cDNA of α_1D-AR subtypes were cultured and cell membrane stationary phase (CMSP) was prepared. Then the interactions between ligands and α_1D-AR in CMSP were investigated using CMC. The affinity rank order to α_1D-AR subtype obtained from CMC for the nine α₁-adrenoceceptor ligands is: prazosin, BMY7378, phentolamine, oxymetazoline, 5-methylurapidil, norepinephrine, phenyle-phrine, methoxamine, RS-17053. The affinity rank order is similar and correlates well with that obtained from others’ radioligand binding assays (RBA). CMSP prepared by transfected HEK293 cells with α_1D-adrenoceptor cDNA and CMC method could be used to evaluate affinities of drug-receptor and drug-receptor subtypes and to screen drugs selective to α_1D-AR.     

Alpha1 adrenoceptor subtypes in human urinary bladder: sex and regional comparison

A detailed study of the presence of alpha1 AR binding sites and alpha1 AR subtype mRNA expression in human urinary bladder areas involved in the micturition (i.e. detrusor, trigone and neck) is reported here, investigating whether or not there are differences between sexes. Results obtained indicated that alpha1 AR proteins were detectable in each bladder area. In both sexes, the detrusor and the neck expressed similar levels of alpha1 ARs: respectively, detrusor: 14.6 +/- 1.2 in men and 13.1 +/- 1.1 fmol/mg prot in women; neck: 16.9 +/- 3.2 in men and 17.5 +/- 4.1 fmol/mg prot in women. In the trigone, significantly higher alpha1ARs were found in women compared to men (20.6 +/- 1.1 vs 11.7 +/- 0.7 fmol/mg prot). Subtype analysis indicated that in women, each area was endowed with mRNA encoding for each alpha1 AR subtype. The men detrusor expressed alpha1a and alpha1d ARs, while in the trigone and the neck, each subtype was present. Since the detrusor muscle hypertrophy is a marker of bladder obstructive outlet, the selective alpha1 AR subtype targeting arouses much interest, as evidence indicates that there are differences in signalling pathways among the subtypes. Furthermore, the significance of the alpha1 ARs coexpression is still unknown; interestingly, recent papers demonstrate that alpha1 AR subtypes could dimerize. Thus, in the human urinary bladder it may be suggested a potential level of alpha1 AR complexity that could have an impact on drug development.     

Alpha-conotoxins ImI and ImII target distinct regions of the human alpha7 nicotinic acetylcholine receptor and distinguish human nicotinic receptor subtypes

The Conus peptides alpha-conotoxin ImI (alpha-ImI) and ImII (alpha-ImII) differ by only three of 11 residues in their primary sequences and yet are shown to inhibit the human alpha7 nicotinic acetylcholine receptor (nAChR) by targeting different sites. Mutations at both faces of the classical ligand binding site of the alpha7 nAChR strongly affect antagonism by alpha-ImI but not alpha-ImII. The effects of the mutations on alpha-ImI binding and functional antagonism are explained by computational docking of the NMR structure of alpha-ImI to a homology model of the ligand binding domain of the alpha7 nAChR. A distinct binding site for alpha-ImII is further demonstrated by its weakened antagonism for a chimeric receptor in which the membrane-spanning domains and intervening linkers of the alpha7 nAChR are replaced with the corresponding sequence from the serotonin type-3 receptor (5HT(3)). The two toxins also discriminate between different subtypes of human nicotinic receptors; alpha-ImII most strongly blocks the human alpha7 and alpha1beta1deltaepsilon receptor subtypes, while alpha-ImI most potently blocks the human alpha3beta2 subtype. Collectively, the data show that while alpha-ImI targets the classical competitive ligand binding site in a subtype selective manner, alpha-ImII is a probe of a novel inhibitory site in homomeric alpha7 nAChRs.     

A new class of RAR subtype selective retinoids: correlation of pharmacological effects with receptor activity

The synthesis and biological activity of a series of structurally related retinoids with different RAR subtype selectivities are described. These retinoids bind to all three RAR subtypes but in functional transactivation assays, they show RARbeta or RARbeta,gamma selectivity with weak RARalpha activity. The subtype selectivity of these retinoids was found to correlate with their efficacy (ODC inhibition) and toxicity (topical irritation and teratogenicity) profiles. The degree of RARgamma transactivation activity correlates with their topical toxicity and teratogenicity as measured by the inhibition of chondrogenesis. Of the RARbeta selective retinoids reported here, retinoid 12 is the most promising, as it is completely devoid of two common retinoid related toxicities, namely topical irritation and teratogenesis.     

Dominance of the alpha1B-adrenergic receptor and its subcellular localization in human and TRAMP prostate cancer cell lines

The function and distribution of alpha1-adrenergic receptor (AR) subtypes in prostate cancer cells is well characterized. Previous studies have used RNA localization or low-avidity antibodies in tissue or cell lines to determine the alpha1-AR subtype and suggested that the alpha1A-AR is dominant. Two androgen-insensitive, human metastatic cancer cell lines DU145 and PC3 were used as well as the mouse TRAMP C1-C3 primary and clonal cell lines. The density of alpha1-ARs was determined by saturation binding and the distribution of the different alpha1-AR subtypes was examined by competition-binding experiments. In contrast to previous studies, the major alpha1-AR subtype in DU145, PC3 and all of the TRAMP cell lines is the alpha1B-AR. DU145 cells contained 100% of the alpha1B-AR subtype, whereas PC3 cells were composed of 21% alpha1 A-AR and 79% alpha1B-AR. TRAMP cell lines contained between 66% and 79% of the alpha1B-AR with minor fractions of the other two subtypes. Faster doubling time in the TRAMP cell lines correlated with decreasing alpha 1B-AR and increasing alpha1 A- and alpha1D-AR densities. Transfection with EGFP-tagged alpha1B-ARs revealed that localization was mainly intracellular, but the majority of the receptors translocated to the cell surface after extended preincubation (18 hr) with either agonist or antagonist. Localization was confirmed by ligand-binding studies and inositol phosphate assays where prolonged preincubation with either agonist and/or antagonist increased the density and function of alpha 1-ARs, suggesting that the native receptors were mostly intracellular and nonfunctional. Our studies indicate that alpha1B-ARs are the major alpha1-AR subtype expressed in DU145, PC3, and all TRAMP cell lines, but most of the receptor is localized in intracellular compartments in a nonfunctional state, which can be rescued upon prolonged incubation with any ligand.     

Comparative expression of the human beta(2) and beta(3) adrenergic receptors in Saccharomyces cerevisiae

The beta(3) adrenergic receptor (beta(3)AR) is the predominant beta subtype in human brown adipocytes and is essential for regulating thermogenic lipolysis. To establish a novel experimental system for the biochemical analysis of this protein, we engineered several yeast strains. We show that the sterol background of the host strain greatly modulates the beta(3)AR expression but not in the same way as it modulates the beta(2) adrenergic receptor (beta(2)AR), the other main studied adipocyte subtype. The human beta(3)AR expressed in yeast is N-glycosylated but not phosphorylated. This latter characteristic distinguishes it from the beta(2)AR. We showed that both beta(2)AR and beta(3)AR follow the secretory pathway to the yeast plasma membrane (PM) and are degraded in the vacuole. In the yeast strains used in this work, the two receptors also share a common mechanism of direct signal transduction through the yeast G(alpha) protein, Gpa1p. These strains thus appear to be useful for biochemical and structural studies of the human beta(3)AR in an in vivo reconstitution system.     

Synthesis of 3,6-diazabicyclo[3.1.1]heptanes as novel ligands for neuronal nicotinic acetylcholine receptors

Alpha series of novel 3,6-diazabicyclo[3.1.1]heptane derivatives 4a-f was synthesized and their affinity and selectivity towards alpha4beta2 and alpha7 nAChR subtypes were evaluated. The results of the current study revealed a number of compounds (4a, 4b and 4c) having a very high affinity for alpha4beta2 (K(i) at alpha4beta2 ranging from 0.023 to 0.056 nM) versus alpha7 nAChR subtypes; among these compounds, the 3-(6-bromopyridin-3-yl)-3,6-diazabicyclo[3.1.1]heptane 4c was found to be the most alpha7alpha4beta2 selective term in receptor binding assays (alpha7alpha4beta2=1295). Moreover, compound 4d also had high affinity for the alpha4beta2 nAChR subtype (K(i)=1.2 nM) with considerably high selectivity (alpha7/alpha4beta2=23300).     

Biochemical properties of sodium channels in a wide range of excitable tissues studied with site-directed antibodies

Antibodies against a peptide (SP19) corresponding to a highly conserved, predicted intracellular region of the sodium channel alpha subunit bind rat brain sodium channels with a similar affinity as the peptide antigen, indicating that the corresponding segment of the alpha subunit is fully accessible in the intact channel structure. These antibodies recognize sodium channel alpha subunits from rat or eel brain, rat skeletal muscle, rat heart, eel electroplax, and locust nervous system. alpha subunits from all these tissues except rat skeletal muscle are substrates for phosphorylation by cAMP-dependent protein kinase. Disulfide linkage of alpha and beta 2 subunits was observed for both the RI and RII subtypes of rat brain sodium channels and for sodium channels from eel brain but not for sodium channels from rat heart, eel electroplax, or locust nerve cord. Treatment with neuraminidase reduced the apparent molecular weight of sodium channel alpha subunits from rat and eel brain and eel electroplax by 22,000-58,000, those from heart by 8000, and those from locust nerve cord by less than 4000. Our results provide the first identification of sodium channel alpha subunits from rat heart and locust brain and nerve cord and show that sodium channel alpha subunits are expressed with different subunit associations and posttranslational modifications in different excitable tissues.     

Triterpenes from Alisma orientalis act as androgen receptor agonists,progesterone receptor antagonists,and glucocorticoid receptor antagonists

Alisma orientalis, a well-known traditional medicine, exerts numerous pharmacological effects including anti-diabetes, anti-hepatitis, and anti-diuretics but its bioactivity is not fully clear. Androgen receptor (AR), progesterone receptor (PR), and glucocorticoid receptor (GR) are three members of nuclear receptor superfamily that has been widely targeted for developing treatments for essential diseases including prostate cancer and breast cancer. In this study, two triterpenes, alisol M 23-acetate and alisol A 23-acetate from Alisma orientalis were determined whether they may act as androgen receptor (AR), progesterone receptor (PR), or glucocorticoid receptor (GR) modulators. Indeed, in the transient transfection reporter assays, alisol M 23-acetate and alisol A 23-acetate transactivated AR in dose-dependent manner, while they transrepressed the transactivation effects exerted by agonist-activated PR and GR. Through molecular modeling docking studies, they were shown to respectively interact with AR, PR, or GR ligand binding pocket fairly well. All these results indicate that alisol M 23-acetate and alisol A 23-acetate from Alisma orientalis might possess therapeutic effects through their modulation of AR, PR, and GR pathways.     

Expression of estrogen receptors in human corpus cavernosum and male urethra.

Estrogen, largely produced in testis and adrenal gland, may play important roles in male reproduction. Most of the effects of estrogens are mediated by binding of estrogen to one or both of the two estrogen receptor (ER) subtypes alpha and beta. Recently, they have been described in testis, prostate, and efferent ducts, mostly in rodents. The goal of this study was to prove the evidence of ERs in human corpus cavernosum and male urethra, exploring the protein expression of these receptors by immunohistochemistry. Corpus cavernosum and corpus spongiosum smooth muscle was immunoreactive for the androgen receptor (AR), ER alpha, and strongly for ER beta. Endothelial cells were negative for AR, sporadically positive for ER alpha, and positive for ER beta. Urethral epithelium showed strong nuclear expression of AR, predominantly in the basal cell layer, and nuclear expression of ER alpha in the intermediate cells. ER beta was highly expressed in almost all urethral nuclei and, much more weakly, in cytoplasm. Progesterone receptor (PGR) was negative in all cases and all tissues. These results represent the first report that ER alpha and particularly ER beta are regularly expressed in human penile tissue.     

Specific recognition of androgens by their nuclear receptor. A structure-function study

Androgens, like progestins, are 3-ketosteroids with structural differences restricted to the 17beta substituent in the steroid D-ring. To better understand the specific recognition of ligands by the human androgen receptor (hAR), a homology model of the ligand-binding domain (LBD) was constructed based on the progesterone receptor LBD crystal structure. Several mutants of residues potentially involved in the specific recognition of ligands in the hAR were constructed and tested for their ability to bind agonists. Their transactivation capacity in response to agonist (R1881) and antagonists (cyproterone acetate, hydroxyflutamide, and ICI 176344) was also measured. Substitution of His(874) by alanine, only marginally impairs the ligand-binding and transactivation capacity of the hAR receptor. In contrast, mutations of Thr(877) and, to a greater extent, Asn(705) perturb ligand recognition, alter transactivation efficiency, and broaden receptor specificity. Interestingly, the N705A mutant acquires progesterone receptor (PR) properties for agonist ligands but, unlike wild type AR and PR, loses the capacity to repress transactivation with nonsteroidal antagonists. Models of the hAR.LBD complexes with several ligands are presented, which suggests new directions for drug design.     

Antidepressants noncompetitively inhibit nicotinic acetylcholine receptor function

Nicotinic acetylcholine receptors (nAChRs) are diverse members of the neurotransmitter-gated ion channel superfamily and play critical roles in chemical signaling throughout the nervous system. The present study establishes for the first time the acute functional effects of sertraline (Zoloft), paroxetine (Paxil), nefazodone (Serzone), and venlafaxine (Effexor) on two human and one chick nAChR subtype. This study also confirms previous findings of nAChR functional block by fluoxetine (Prozac). Function of human muscle-type nAChR (alpha1/beta gammadelta) in TE671/RD cells, human autonomic nAChR (alpha3/beta4alpha5 +/- beta2) in SH-SY5Y neuroblastoma cells, or chick V274T mutant alpha7-nAChR heterologously expressed in native nAChR-null SH-EP1 epithelial cells was measured using 86Rb+ efflux assays. Functional blockade of human muscle-type and autonomic nAChRs is produced by each of the drugs in the low to intermediate micromolar range, and functional blockade of chick V274T-alpha7-nAChR is produced in the intermediate to high micromolar range. Functional blockade is insurmountable by increasing agonist concentrations at each nAChR subtype tested for each of these drugs, suggesting noncompetitive inhibition of nAChR function. These studies open the possibilities that nAChR subtypes in the brain could be targets for therapeutic antidepressants and could play roles in clinical depression.     

Dienogest is a selective progesterone receptor agonist in transactivation analysis with potent oral endometrial activity due to its efficient pharmacokinetic profile

Dienogest was introduced as an oral progestin. Yet its strong oral potency on endometrial activity is not clearly explained. To circumvent this situation, steroid hormone receptor profiling using transactivation assay and endometrial activity test in rabbits were carried out with determination of plasma drug concentration. Agonistic/antagonistic activity on human progesterone receptor (PR), androgen receptor (AR), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), estrogen receptor alpha (ERalpha), or estrogen receptor beta (ERbeta) were determined. Dienogest activate PR (EC50=3.4 or 10.5 nmol/l) with antagonistic activity on AR (EC50=420.6 or 775.0 nmol/l) but not agonistic nor antagonistic action on GR, MR (3000 nmol/l). Dienogest activate neither ERalpha nor ERbeta (3000 nmol/l). Progesterone activated PR with antagonistic activity on AR and on MR. Dydrogesterone showed a similar profile to progesterone. Norethisterone activated PR, AR, and ERalpha. Medroxyprogesterone acetate activated PR, AR, and GR. Danazol activated PR and AR. Collectively, dienogest has a good specificity to PR compared with the other drugs. By oral treatment, dienogest showed the strongest endometrial activity (ED50=0.0042 mg/kg) in McPhail test among other progestins (ED50 values for MPA, DYG, NES were 0.074, 1.9, >0.05 mg/kg, respectively). Dienogest showed higher plasma concentrations than those of the other progestins with higher doses. The estimated plasma concentration of dienogest at ED50 (3.66 nmol/l) was close to its EC50 value to activate PR. Thus, the stronger oral activity of dienogest could be explained simply by its in vitro potency on PR and its oral pharmacokinetic profile.