A Melanophore-Based Screening Assay for Erythropoietin Receptors

A rapid, functional assay in frog melanophore cells for the erythropoietin receptor (EPOR), a member of the cytokine receptor family, is demonstrated. A chimeric receptor that comprised the extracellular portion of the murine EPOR and the transmembrane and intracellular domains of the human epidermal growth factor receptor (EGFR) was subcloned into the expression vector pJG3.6. When the full-length EGFR was expressed in melanophores, EGF but not EPO mediated pigment dispersion in a time- and dose-dependent manner with an EC50 of 12.6 6 2.9 pM. However, when the chimeric EPOR/EGFR was expressed, EPO but not EGF stimulated pigment dispersion in a time- and dose-dependent manner with an EC50 of 380 6 107 pM. Neither EGF nor EPO had any effect on pigment dispersion in wild-type melanophores. EGF- and EPO-mediated pigment dispersion was blocked by the bis-indolylmaleimide protein kinase C inhibitor Ro 31-8220. This study extends the use of the melanophore-based bioassay to include cytokine receptors in addition to G protein- and tyrosine kinase-coupled receptors. It represents a potentially powerful method for screening of combinatorial libraries to identify novel small molecule agonists and antagonists to this clinically important class of binding sites as well as performing studies of functional ligand-receptor interactions.     

A rapid quantitative bioassay for evaluating the effects of ligands upon receptors that modulate cAMP levels in a melanophore cell line.

A new method for rapidly evaluating the effects of drugs on receptors that regulate intracellular cAMP in a cell line derived from Xenopus laevis melanophores has been developed. Melanophores were plated into sterile 96 well microtiter plates, and 3 days later the cells were treated with melatonin for 30 min to induce melanosome aggregation. Subsequent exposure to MSH or adrenergic agonists caused dose dependent pigment dispersion that peaked within 30 min. The cumulative pigment displacement from cells could be quantitated by using a microplate reader to measure changes in transmittance of light through the wells. The acquired data enabled detailed and reproducible dose response curves and time course analyses to be generated. In addition, the assay followed for the rapid characterization of the effects of antagonists upon the beta adrenergic receptor (beta AR). The assay has the potential to test the effects of ligands upon any receptor capable of mediating pigment translocation in the melanophore cell line.     

A Rapid Quantitative Bioassay for Evaluating the Effects of Ligands Upon Receptors That Modulate cAMP Levels in a Melanophore Cell Line

A new method for rapidly evaluating the effects of drugs on receptors that regulate intracellular cAMP in a cell line derived from Xenopus laevis melanophores has been developed. Melanophores were plated into sterile 96 well microtiter plates, and 3 days later the cells were treated with melatonin for 30 min to induce melanosome aggregation. Subsequent exposure to MSH or adrenergic agonists caused dose dependent pigment dispersion that peaked within 30 min. The cumulative pigment displacement from cells could be quantitated by using a microplate reader to measure changes in transmittance of light through the wells. The acquired data enabled detailed and reproducible dose response curves and time course analyses to be generated. In addition, the assay followed for the rapid characterization of the effects of antagonists upon the (β adrenergic receptor (β AR). The assay has the potential to test the effects of ligands upon any receptor capable of mediating pigment translocation in the melanophore cell line.     

Fluorescent localization of the beta-adrenergic receptor on DDT-1 cells. Down-regulation by adrenergic agonists.

Continuous incubation of cultured cells with beta-adrenergic agonists results in the desensitization of adrenergic responsiveness accompanied by the down-regulation of cell surface beta-adrenergic receptors (beta AR). Previous studies have relied on measurements of ligand binding activity for the detection of the beta AR in the cell. In the present study, we have raised a monoclonal antibody to a synthetic peptide corresponding to amino acid numbers 226-239 of the hamster beta 2AR. This antibody was used to localize the beta AR in hamster smooth-muscle DDT-1 cells by immunofluorescence, without regard for the ability of the receptor to bind ligands. The beta AR was found to be localized primarily at the plasma membrane of these cells, with a nonhomogeneous pattern of distribution. A rapid loss of beta AR-specific immunofluorescence, which paralleled receptor down-regulation as measured by ligand-binding activity, was seen with beta-adrenergic agonists, but not with antagonists. In addition, a transient increase in fluorescence was observed after short times of exposure of the cells to agonists. This fluorescence increase may reflect a ligand-induced conformational change in the receptor.     

Functional characterization of a receptor for vasoactive-intestinal-peptide-related peptides in cultured dermal melanophores from Xenopus laevis

A receptor for vasoactive-intestinal-peptide (VIP)-related peptides was functionally characterized in a cell line derived from Xenopus melanophores using a recently described microtiter-plate-based bioassay. Activation of the melanophore VIP receptor by VIP or the peptides pituitary-adenylate-cyclase-activating polypeptide (PACAP 38), PACAP 27, and helodermin stimulated intracellular 3'-5' cyclic adenosine monophosphate (cAMP) accumulation and pigment dispersion in the cells. Helodermin, with an EC50 (concentration of peptide inducing half-maximal melanosome dispersion) of 46.5 pM, was the most potent activator of pigment dispersion, followed by PACAP 38 > VIP > PACAP 27. A similar order of potencies was observed for the peptides to induce cAMP accumulation. The responses to VIP agonists were selectively inhibited by the VIP antagonists PACAP-(6-27) and (N-Ac-Tyr(1)-D-Phe2)-growth-hormone-releasing factor[GRF](1-29)-NH2. Taken together, the results suggest that the melanophores express a VIP receptor that shares certain characteristics of, but also differs significantly from, other previously identified VIP receptors.     

alpha 2B-Adrenoceptor levels govern agonist and inverse agonist responses in PC12 cells

Receptor density is an important determinant of cellular effector responses to receptor activation. We analysed cytosolic Ca(2+) responses to alpha(2)-adrenergic agents in PC12 cells expressing human alpha(2B)-adrenergic receptors (AR) at two densities (3.8 and 1.3 pmol/mg protein). The efficacy (E(max)) of agonists was greater in cells with higher receptor expression; while the potency (EC(50)) of norepinephrine and oxymetazoline was independent of alpha(2B)-AR levels. Several classical alpha(2)-AR antagonists behaved as either partial or inverse agonists in a receptor density-dependent fashion. No apparent structural similarities were found among the inverse agonists, precluding simple predictions of inverse agonist activity. Transfected PC12 cells expressing alpha(2B)-AR at relatively high density would be a useful approach to screen inverse agonists for this class of receptors. Our results further indicate that receptor density significantly influences the properties of ligands, not only of partial agonists as predicted by classical receptor theory, but also of antagonists and full agonists.     

Functional coupling of the Galpha(olf) variant XLGalpha(olf) with the human adenosine A2A receptor

A recently identified novel Galphaolf variant, XLGalphaolf, is shown to functionally couple to the human adenosine A2A receptor (A2AR). In Sf9 cells expressing A2AR, beta1, and gamma2, co-expression of XLGalphaolf increased NECA-induced [35S]GTPgammaS binding from approximately 130% to 300% of basal levels. Pharmacological characteristics of A2AR ligands on these cells were evaluated by using [3H]ZM241385- and [35S]GTPgammaS- binding assays. The rank order of the equilibrium binding constants (Kd or Ki) of adenosine receptor ligands were [3H]ZM241385 approximately CGS15943 < MRS1220 < < CV1808 approximately NECA < CGS21680 approximately adenosine < IBMECA < HEMADO approximately CPA approximately CCPA. The rank order of EC50 values for agonists were CV1808 approximately NECA < adenosine approximately CGS26180 < IBMECA < HEMADO approximately CPA approximately CCPA. This pharmacology is consistent with the literature for A2AR and suggests that Sf9 cells co-expressing A2AR, beta1, gamma2, and XLGalphaolf could serve as a heterologous expression system for A2AR drug screening.     

Functional assays to define agonists and antagonists of the sigma-2 receptor

The sigma-2 receptor has been identified as a biomarker in proliferating tumors. To date there is no well-established functional assay for defining sigma-2 agonists and antagonists. Many sigma-2 ligands with diverse structures have been shown to induce cell death in a variety of cancer cells by triggering caspase-dependent and independent apoptosis. Therefore, in the current study, we used the cell viability assay and the caspase-3 activity assay to determine sigma-2 agonists and antagonists. Three classes of sigma-2 ligands developed in our laboratory were evaluated for their potency to induce cell death in two tumor cell lines, mouse breast cancer cell line EMT-6 and human melanoma cell line MDA-MB-435. The data showed that the EC₅₀ values of the sigma-2 ligands using the cell viability assay ranged from 11.4 μM to >200 μM, which were comparable with the EC₅₀ values obtained using the caspase-3 assay. Based on the cytotoxicity of a sigma-2 ligand relative to that of siramesine, a commonly accepted sigma-2 agonist, we have categorized our sigma-2 ligands into agonists, partial agonists, and antagonists. The establishment of functional assays for defining sigma-2 agonists and antagonists will facilitate functional characterization of sigma-2 receptor ligands and sigma-2 receptors.     

Antiproliferative effects of selective adenosine receptor agonists and antagonists on human lymphocytes: evidence for receptor-independent mechanisms

The effects of standard adenosine receptor (AR) agonists and antagonists on the proliferation of human T lymphocytes, unstimulated and phytohemagglutinin-stimulated human peripheral blood lymphocytes (PBL), and Jurkat T cells were investigated. Real-time PCR measurements confirmed the presence of all four AR subtypes on the investigated cells, although at different expression levels. A_2A ARs were predominantly expressed in PBL and further upregulated upon stimulation, while malignant Jurkat T cells showed high expression levels of A₁, A_2A, and A_2B ARs. Cell proliferation was measured by [³H]-thymidine incorporation assays. Several ligands, including the subtype-selective agonists CPA (A₁), BAY60-6583 (A_2B), and IB-MECA (A₃), and the antagonists PSB-36 (A₁), MSX-2 (A_2A), and PSB-10 (A₃) significantly inhibited cell proliferation at micromolar concentrations, which were about three orders of magnitude higher than their AR affinities. In contrast, further investigated AR ligands, including the agonists NECA (nonselective) and CGS21680 (A_2A), and the antagonists preladenant (SCH-420814, A_2A), PSB-1115 (A_2B), and PSB-603 (A_2B) showed no or only minor effects on lymphocyte proliferation. The anti-proliferative effects of the AR agonists could not be blocked by the corresponding antagonists. The non-selective AR antagonist caffeine stimulated phytohemagglutinin-activated PBL with an EC₅₀ value of 104 μM. This is the first study to compare a complete set of commonly used AR ligands for all subtypes on lymphocyte proliferation. Our results strongly suggest that these compounds induce an inhibition of lymphocyte proliferation and cell death through AR-independent mechanisms.     

beta-arrestin-biased agonism at the beta2-adrenergic receptor

Classically, the beta 2-adrenergic receptor (beta 2AR) and other members of the seven-transmembrane receptor (7TMR) superfamily activate G protein-dependent signaling pathways in response to ligand stimulus. It has recently been discovered, however, that a number of 7TMRs, including beta 2AR, can signal via beta-arrestin-dependent pathways independent of G protein activation. It is currently unclear if among beta 2AR agonists there exist ligands that disproportionately signal via G proteins or beta-arrestins and are hence "biased." Using a variety of approaches that include highly sensitive fluorescence resonance energy transfer-based methodologies, including a novel assay for receptor internalization, we show that the majority of known beta 2AR agonists exhibit relative efficacies for beta-arrestin-associated activities (beta-arrestin membrane translocation and beta 2AR internalization) identical to the irrelative efficacies for G protein-dependent signaling (cyclic AMP generation). However, for three betaAR ligands there is a marked bias toward beta-arrestin signaling; these ligands stimulate beta-arrestin-dependent receptor activities to a much greater extent than would be expected given their efficacy for G protein-dependent activity. Structural comparison of these biased ligands reveals that all three are catecholamines containing an ethyl substitution on the alpha-carbon, a motif absent on all of the other, unbiased ligands tested. Thus, these studies demonstrate the potential for developing a novel class of 7TMR ligands with a distinct bias for beta-arrestin-mediated signaling.     

Identification of an allosteric binding site for Zn2+ on the beta2 adrenergic receptor

The activity of G protein-coupled receptors (GPCRs) can be modulated by a diverse spectrum of drugs ranging from full agonists to partial agonists, antagonists, and inverse agonists. The vast majority of these ligands compete with native ligands for binding to orthosteric binding sites. Allosteric ligands have also been described for a number of GPCRs. However, little is known about the mechanism by which these ligands modulate the affinity of receptors for orthosteric ligands. We have previously reported that Zn(II) acts as a positive allosteric modulator of the beta(2)-adrenergic receptor (beta(2)AR). To identify the Zn(2+) binding site responsible for the enhancement of agonist affinity in the beta(2)AR, we mutated histidines located in hydrophilic sequences bridging the seven transmembrane domains. Mutation of His-269 abolished the effect of Zn(2+) on agonist affinity. Mutations of other histidines had no effect on agonist affinity. Further mutagenesis of residues adjacent to His-269 demonstrated that Cys-265 and Glu-225 are also required to achieve the full allosteric effect of Zn(2+) on agonist binding. Our results suggest that bridging of the cytoplasmic extensions of TM5 and TM6 by Zn(2+) facilitates agonist binding. These results are in agreement with recent biophysical studies demonstrating that agonist binding leads to movement of TM6 relative to TM5.     

Human beta3 adrenergic receptor agonists containing cyanoguanidine and nitroethylenediamine moieties

Pyridineethanolamine derivatives containing cyanoguanidine or nitroethylenediamine moieties were examined as human beta3 adrenergic receptor (AR) agonists. Notably, indoline derivatives 6a and 11 were potent beta3 AR agonists (beta3 EC50 = 13 and 19 nM, respectively), which showed good selectivity over binding to and minimal activation of the beta1 and beta2 ARs.     

Recent developments in adenosine receptor ligands and their potential as novel drugs

Medicinal chemical approaches have been applied to all four of the adenosine receptor (AR) subtypes (A(1), A(2A), A(2B), and A(3)) to create selective agonists and antagonists for each. The most recent class of selective AR ligands to be reported is the class of A(2B)AR agonists. The availability of these selective ligands has facilitated research on therapeutic applications of modulating the ARs and in some cases has provided clinical candidates. Prodrug approaches have been developed which improve the bioavailability of the drugs, reduce side-effects, and/or may lead to site-selective effects. The A(2A) agonist regadenoson (Lexiscan?), a diagnostic drug for myocardial perfusion imaging, is the first selective AR agonist to be approved. Other selective agonists and antagonists are or were undergoing clinical trials for a broad range of indications, including capadenoson and tecadenoson (A(1) agonists) for atrial fibrillation, or paroxysmal supraventricular tachycardia, respectively, apadenoson and binodenoson (A(2A) agonists) for myocardial perfusion imaging, preladenant (A(2A) antagonist) for the treatment of Parkinson's disease, and CF101 and CF102 (A(3) agonists) for inflammatory diseases and cancer, respectively.     

Inhibition of human airway smooth muscle cell proliferation by beta 2-adrenergic receptors and cAMP is PKA independent: evidence for EPAC involvement

Mechanisms by which beta-adrenergic receptor (beta AR) agonists inhibit proliferation of human airway smooth muscle (HASM) cells were investigated because of their potential relevance to smooth muscle hyperplasia in asthma. We hypothesized that beta AR agonists would inhibit mitogenesis in HASM cells via the beta 2AR, an increase in cAMP, and PKA activation. HASM cells were treated for 24 h with various agents and then analyzed for [3H]thymidine incorporation as a measure of cell proliferation. EGF stimulated proliferation by approximately 10-fold. The nonselective beta AR agonist isoproterenol and the beta 2AR-selective agonists albuterol and salmeterol inhibited EGF-stimulated proliferation by more than 50%, with half-maximal effects at 4.8 nM, 110 nM, and 6.7 nM, respectively. A beta 2AR-selective antagonist inhibited the isoproterenol effect with 100-fold greater potency than a beta 1AR-selective antagonist, confirming beta 2AR involvement in the inhibition of proliferation. The cAMP-elevating agents PGE2 and forskolin decreased EGF-induced proliferation, suggesting cAMP as the mediator. beta 2AR agonists and forskolin also inhibited proliferation stimulated by lysophosphatidic acid (LPA) as well as the synergistic proliferation stimulated by LPA+EGF. Importantly, PKA-selective cAMP analogs did not inhibit proliferation at concentrations that maximally activated PKA (10-100 microM), whereas a cAMP analog selective for the exchange protein directly activated by cAMP (EPAC), 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, maximally inhibited proliferation at a concentration that did not activate PKA (10 microM). These data show that beta 2AR agonists and other cAMP-elevating agents decrease proliferation in HASM cells via a PKA-independent mechanism, and they provide pharmacological evidence for involvement of EPAC or an EPAC-like cAMP effector protein instead.     

Antidepressant-induced switch of beta 1-adrenoceptor trafficking as a mechanism for drug action

Reduction in surface beta(1)-adrenoceptor (beta1AR) density is thought to play a critical role in mediating the therapeutic long term effects of antidepressants. Since antidepressants are neither agonists nor antagonists for G protein-coupled receptors, receptor density must be regulated through processes independent of direct receptor activation. Endocytosis and recycling of the beta1AR fused to green fluorescent protein at its carboxyl-terminus (beta1AR-GFP) were analyzed by confocal fluorescence microscopy of live cells and complementary ligand binding studies. In stably transfected C6 glioblastoma cells, beta1AR-GFP displayed identical ligand-binding isotherms and adenylyl cyclase activation as native beta1AR. Upon exposure to isoproterenol, a fraction of beta1AR-GFP (10-15%) internalized rapidly and colocalized with endocytosed transferrin receptors in an early endosomal compartment in the perinuclear region. Chronic treatment with the tricyclic antidepressant desipramine (DMI) did not affect internalization characteristics of beta1AR-GFP when challenged with isoproterenol. However, internalized receptors were not able to recycle back to the cell surface in DMI-treated cells, whereas recycling of transferrin receptors was not affected. Endocytosed receptors were absent from structures that stained with fluorescently labeled dextran, and inhibition of lysosomal protease activity did not restore receptor recycling, indicating that beta1AR-GFP did not immediately enter the lysosomal compartment. The data suggest a new mode of drug action causing a "switch" of receptor fate from a fast recycling pathway to a slowly exchanging perinuclear compartment. Antidepressant-induced reduction of receptor surface expression may thus be caused by modulation of receptor trafficking routes.     

Coexistence of beta 1- and beta 2-adrenoceptors in the melanophore of the goby Tridentiger obscurus

The effects of beta-adrenergic agonists and antagonists on the pigmentary state of denervated melanophores in isolated, split, caudal fins of the goby Tridentiger obscurus were examined to investigate the function and the subtype of the beta-adrenoceptors of the melanophores. Salbutamol, terbutaline, and dobutamine partially inhibited the pigment-aggregating response of melanophores to norepinephrine. The effects of these beta-agonists were inhibited by propranolol. It was confirmed that the melanophores possess both alpha- and beta-adrenoceptors, and that the activation of the beta-adrenoceptors induces the dispersion of pigment in the melanophores. Norepinephrine, epinephrine, isoproterenol, dobutamine, salbutamol, and terbutaline evoked the dispersion of pigment in the melanophores in which pigment had previously been aggregated by treatment with verapamil in the presence of phentolamine. The pigment-dispersing effects of two beta 1-selective agonists, norepinephrine and dobutamine, were effectively inhibited by metoprolol, a selective antagonist of beta 1-receptors. By contrast, the pigment-dispersing effects of two beta 2-selective agonists, salbutamol and terbutaline, were not inhibited by metoprolol. Both the effects of nonselective agonists, epinephrine and isoproterenol, were partially inhibited by metoprolol. The actions of all of the beta-agonists used were effectively inhibited by propranolol, and they were partially inhibited by butoxamine. These results suggest co-existence of beta 1- and beta 2-adrenoceptors in the melanophores. The relative numbers of beta 1- and beta 2-adrenoreceptors as a percentage of the total population of beta-adrenoceptors were estimated to be 18.6% and 81.4%, respectively, from analyses of Hofstee plots of the effects of the beta-agonists on the melanophores in the presence of butoxamine or metoprolol.     

Allele-specific activation of genetically engineered receptors.

The binding of agonists and antagonists to the beta-adrenergic receptor (beta AR) is postulated to involve an ionic interaction between the amine group of the ligand and the carboxylate side chain of Asp113 in the third hydrophobic domain of the receptor. To explore the importance of this interaction in the binding of ligands to the beta AR, a Ser residue was substituted for Asp113, and the ability of this mutant receptor to respond to compounds which could potentially interact with the hydroxyl side chain of the Ser residue was assessed. The mutant receptor was fully activated by catechol-containing esters and ketones, compounds which did not activate the wild-type beta AR. The demonstration that the molecular substitution of a single amino acid residue can alter the ligand binding specificity of the beta AR provides evidence that the chemical nature of this residue is a critical determinant in the recognition site of the receptor. Further, the ability to modify the specificity of a receptor by the replacement of amino acids at the binding site demonstrates the potential for the rational design of drugs which function specifically at genetically engineered receptors.     

Expression of three human beta-adrenergic-receptor subtypes in transfected Chinese hamster ovary cells.

The genes coding for three pharmacologically distinct subtypes of human beta-adrenergic receptors (beta 1 AR, beta 2 AR and beta 3 AR) were transfected for expression in Chinese hamster ovary (CHO) cells. Stable cell lines expressing each receptor were analyzed by ligand binding, adenylate cyclase activation and photoaffinity labeling, and compared to beta AR subtypes observed in previously described tissues, primary cultures and transfected cell lines. Each of the three receptor subtypes displayed saturable [125I]iodocyanopindolol-binding activity. They showed the characteristic rank order of potencies for five agonists, determined by measuring the accumulation of intracellular cAMP. These recombinant cell lines express a homogeneous population of receptors and display the known pharmacological properties of beta 1 AR and beta 2 AR, in human tissues. It is therefore likely that the pattern of ligand binding and adenylate cyclase activation, mediated by the new beta 3 AR in CHO cells, also reflects the yet-undetermined pharmacological profile in humans.     

Structural basis for androgen receptor agonists and antagonists: interaction of SPEED 98-listed chemicals and related compounds with the androgen receptor based on an in vitro reporter gene assay and 3D-QSAR

The androgen receptor (AR) activity of listed chemicals, so called SPEED 98, by the Ministry of the Environment, Japan, and structurally related chemicals was characterized using MDA-kb2 human breast cancer cells stably expressing an androgen-responsive luciferase reporter gene, MMTV-luc. Since our results suggested that chemicals with diverse chemical structures were capable of disrupting the endocrine systems mediated by AR, a comparative molecular field analysis (CoMFA) model was developed to analyze the structural requirements necessary to disrupt AR function. A significant CoMFA model with r(2)=0.825 and q(2)=0.332 was developed for AR antagonist activity of 35 pure antagonists excluding procymidone. On the other hand, a good CoMFA model with r(2)=0.983 and q(2)=0.555 was obtained for antagonist activity of 13 chemicals with both agonist and antagonist activities. The steric and electrostatic properties were sufficient to describe the structural requirements for AR antagonist activity. In addition, the structural difference of AR agonists and antagonists was explained based on CoMFA results and the AR-LBD crystal structure. As several ERalpha agonists such as diethylstilbestrol (DES) acted as AR antagonists, the surface area of the AR ligand-binding domain (LBD) was compared with that of the ERalpha-LBD based on their reported crystal structures to analyze how those ligands interact with LBDs. The surface area of AR-LBD was shown to be smaller than that of ERalpha-LBD and therefore compounds with both estrogenic and antiandrogenic activities can fit well into the ERalpha-LBD but may protrude from the AR-LBD. It is likely that this subtle difference of the surface areas of the LBDs determines whether an ERalpha agonist acts as an AR antagonist or an agonist.