Radiolabeled Ligand Binding Research Articles

New characterization of dihydroergotamine receptor pharmacology in the context of migraine: utilization of a β-arrestin recruitment assay

IntroductionDihydroergotamine mesylate (DHE) is an established effective acute therapy for migraine and is often characterized by its broad receptor pharmacology. Knowledge of DHE pharmacology largely comes from studies employing older methodologies.ObjectiveTo assess DHE receptor activity using high-throughput methods to screen for functional ß-arrestin activity at G protein–coupled receptors (GPCRs).MethodsFunctional receptor activities of DHE and sumatriptan succinate (both 10 μM) were screened against 168 GPCRs using the gpcrMAX assay. Agonist and antagonist effects were considered significant if receptor activity was >30% or inhibited by >50%, respectively. Radiolabeled ligand binding assays were performed for DHE (0.01–300 nM for 5-HT3 and 4E; 0.3–10,000 nM for 5-HT1B, α-adrenergic2B [i.e., α2B-adrenoceptor], D2, and D5) to assess specific binding to select receptors.ResultsDHE (10 μM) exhibited agonist activity at α-adrenergic2B, CXC chemokine receptor 7 (CXCR7), dopamine (D)2/5, and 5-hydroxytryptamine (5-HT)1A/1B/2A/2C/5A receptors and antagonist activity at α-adrenergic1B/2A/2C (i.e., α1B/2A/2C-adrenoceptors), calcitonin receptor–receptor activity modifying protein 2 (CTR-RAMP2) or amylin 2 (AMY2), D1/3/4/5, and 5-HT1F receptors. Sumatriptan succinate (10 μM) exhibited agonist activity at the 5-HT1B/1E/1F/5A receptors. DHE demonstrated a half-maximal inhibitory concentration (IC50) of 149 nM at the 5-HT1F receptor and a half-maximal effective concentration (EC50) of 6 μM at the CXCR7 receptor. DHE did not bind to the 5-HT3 receptor at concentrations up to 300 nM and bound poorly to 5-HT4E and D5 receptors (IC50 of 230 and 370 nM, respectively). DHE bound strongly to the D2, 5-HT1B, and α-adrenergic2B receptors (IC50 of 0.47, 0.58, and 2.8 nM, respectively).ConclusionBy using a high-throughput β-arrestin recruitment assay, this study confirmed the broad receptor profile of DHE and provided an update on DHE receptor pharmacology as it relates to migraine.

5-HT3 Receptor MX Helix Contributes to Receptor Function.

5-HT3 receptors are members of the family of pentameric ligand-gated ion channels. Each subunit has an extracellular, transmembrane, and intracellular domain. Only part of the intracellular domain structure has been solved, revealing it contains two α-helical segments; one, the MA helix, is an extension of M4, while the other, the MX helix, is formed from residues located close to the end of M3. This MX helix is in distinct locations in open and closed receptor structures, suggesting it may play a role in function. Here, we explore this hypothesis using functional responses of Ala-substituted mutant receptors expressed in HEK293 cells. The data show altering many of the MX residues results in a small decrease in EC50 (up to 5-fold), although in one (H232A) this is increased. Radiolabeled ligand binding on selected mutants showed no change in binding affinity, indicating an effect on gating and not binding. In addition, five mutations (P316A, V317A, P318A, D319A, and H323A) initially resulted in nonfunctional receptors, but the function could be rescued by coexpression with a chaperone protein, suggesting a likely role in assembly or folding. Examination of previously obtained MD simulation data shows that the extent of MX encompassed by membrane lipids differs considerably in the open and closed structures, suggesting that lipid–protein interactions in this region could have a major effect on channel opening propensity. We conclude that the MX helix can modulate the function of the receptor and propose that its interactions with membrane lipids play a major role in this.

Discovery of Novel Allosteric Modulators Targeting an Extra-Helical Binding Site of GLP-1R Using Structure- and Ligand-Based Virtual Screening.

Allosteric modulators have emerged with many potential pharmacological advantages as they do not compete the binding of agonist or antagonist to the orthosteric sites but ultimately affect downstream signaling. To identify allosteric modulators targeting an extra-helical binding site of the glucagon-like peptide-1 receptor (GLP-1R) within the membrane environment, the following two computational approaches were applied: structure-based virtual screening with consideration of lipid contacts and ligand-based virtual screening with the maintenance of specific allosteric pocket residue interactions. Verified by radiolabeled ligand binding and cAMP accumulation experiments, two negative allosteric modulators and seven positive allosteric modulators were discovered using structure-based and ligand-based virtual screening methods, respectively. The computational approach presented here could possibly be used to discover allosteric modulators of other G protein-coupled receptors.

1, 6-di-O-caffeoyl-β-D-glucopyranoside, a natural compound from Callicarpa nudiflora Hook impairs P2Y12 and thromboxane A2 receptor-mediated amplification of platelet activation and aggregation

BackgroundPlatelet activation and subsequent accumulation at sites of vascular injury perform a central role in thrombus formation, which is believed to be the trigger of several cardiovascular diseases, such as atherosclerosis, myocardial infarction and strokes. In this sense, the search for agents that are capable of blocking platelets aggregation has important implications for these diseases. Callicarpa nudiflora (C. nudiflora) Hook is a traditional Chinese medicine herb for eliminating stasis to subdue swelling and hemostasis. Our previous study found several compounds extracted from this herb, including 1, 6-di-O-caffeoyl-β-D-glucopyranoside (CGP), showed inhibitory effects on adenosine diphosphate (ADP) induced platelet aggregation. PurposeThe aim of current study is confirmation of the anti-platelet effects and elucidation of the probable mechanisms. MethodsThe experiments were performed on platelet rich plasma freshly isolated from SD rat. ADP, U46619 or arachidonic acid (AA) induced platelet aggregation assay were performed to evaluate the anti-platelet properties of CGP. Activated αIIbβ3 integrin abundance, serotonin (5-HT) secretion, thromboxane A2 (TXA2) synthesis was determined to assess the effects of CGP on platelet activation. Furthermore, RhoA and PI3K/Akt/GSK3β signal transduction were analyzed by Western Blotting assay. In addition, radiolabelled ligand binding assay was involved to evaluate the ability of CGP binding to thromboxane prostanoid (TP) and P2Y12 receptors. ResultsCGP inhibited platelet aggregation induced by ADP, U46619 and arachidonic acid (AA), significantly. Furthermore, it is also found that LGP exhibited obvious inhibitory effects on αIIbβ3 integrin activation, serotonin (5-HT) secretion from granule and thromboxane A2 (TXA2) synthesis. Next, we found that CGP suppressed RhoA and PI3K/Akt/GSK3β signal transduction. Data from radiolabelled ligand binding assay showed that CGP displayed apparent competing effects on TP and P2Y12 receptors. ConclusionCollectively, the data presented here demonstrated that CGP, a natural compound from Callicarpa nudiflora Hook, inhibited the development of platelet aggregation and amplification of platelet activation. These inhibitory effects may be associated with its dual-receptor inhibition on P2Y12 and TP receptors.

The angiotensin receptor blocker, Losartan, inhibits mammary tumor development and progression to invasive carcinoma

Drugs that target the Renin-Angiotensin System (RAS) have recently come into focus for their potential utility as cancer treatments. The use of Angiotensin Receptor Blockers (ARBs) and Angiotensin-Converting Enzyme (ACE) Inhibitors (ACEIs) to manage hypertension in cancer patients is correlated with improved survival outcomes for renal, prostate, breast and small cell lung cancer. Previous studies demonstrate that the Angiotensin Receptor Type I (AT1R) is linked to breast cancer pathogenesis, with unbiased analysis of gene-expression studies identifying significant up-regulation of AGTR1, the gene encoding AT1R in ER+ve/HER2−ve tumors correlating with poor prognosis. However, there is no evidence, so far, of the functional contribution of AT1R to breast tumorigenesis. We explored the potential therapeutic benefit of ARB in a carcinogen-induced mouse model of breast cancer and clarified the mechanisms associated with its success.Mammary tumors were induced with 7,12-dimethylbenz[α]antracene (DMBA) and medroxyprogesterone acetate (MPA) in female wild type mice and the effects of the ARB, Losartan treatment assessed in a preventative setting (n = 15 per group). Tumor histopathology was characterised by immunohistochemistry, real-time qPCR to detect gene expression signatures, and tumor cytokine levels measured with quantitative bioplex assays. AT1R was detected with radiolabelled ligand binding assays in fresh frozen tumor samples.We showed that therapeutic inhibition of AT1R, with Losartan, resulted in a significant reduction in tumor burden; and no mammary tumor incidence in 20% of animals. We observed a significant reduction in tumor progression from DCIS to invasive cancer with Losartan treatment. This was associated with reduced tumor cell proliferation and a significant reduction in IL-6, pSTAT3 and TNFα levels. Analysis of tumor immune cell infiltrates, however, demonstrated no significant differences in the recruitment of lymphocytes or tumour-associated macrophages in Losartan or vehicle-treated mammary tumors.Analysis of AT1R expression with radiolabelled ligand binding assays in human breast cancer biopsies showed high AT1R levels in 30% of invasive ductal carcinomas analysed. Furthermore, analysis of the TCGA database identified that high AT1R expression to be associated with luminal breast cancer subtype.Our in vivo data and analysis of human invasive ductal carcinoma samples identify the AT1R is a potential therapeutic target in breast cancer, with the availability of a range of well-tolerated inhibitors currently used in clinics. We describe a novel signalling pathway critical in breast tumorigenesis, that may provide new therapeutic avenues to complement current treatments.

Modulation of alfa-adrenoceptor activity by beta-adrenoceptor agonists and antagonists in rat brain cortex membranes

The influence of β-adrenoceptor activation and inhibition by isoprenaline and propranolol on the specific binding of nonselective α1- and α2-adrenoceptor antagonists [3H]prazosin and [3H]RX821002 in rat cerebral cortex subcellular membrane fractions was studied. It was established that for the α1- and α2-adrenoceptors the ligand–receptor interaction corresponds to the model of one affinity pool of receptors and binding of two ligand molecules by one dimer receptor. The parameters of [3H]prazosin binding to α1-adrenoceptors were: K d = 1.85 ± 0.16 nM, B max = 31.14 ± 0.35 fmol/mg protein, n = 2. The parameters of [3H]RX821002 binding to α2-adrenoceptors were: K d = 1.57 ± 0.27 nM, B max = 7.2 ± 1.6 fmol/mg protein, n = 2. When β-adrenoceptors were activated by isoprenaline, the binding of radiolabelled ligands with α1- and α2-adrenoceptors occurred according to the same model. The affinity to [3H]prazosin and the concentration of active α1-adrenoceptors increased by 27% (K d = 1.36 ± 0.03 nM) and 84% (B max = 57.37 ± 0.28 fmol/mg protein), respectively. The affinity of α2-adrenoceptors to [3H]RX821002 decreased by 56% (K d = 3.55 ± 0.02 nM), and the concentration of active receptors increased by 69% (B max = 12.24 ± 0.06 fmol/mg protein). Propranolol alters the binding character of both ligands. For [3H]prazosin and [3H]RX821002, two pools of receptors were detected with the following parameters: K d1 = 1.13 ± 0.09, K d2 = 6.07 ± 1.06 nM, B m1 = 11.36 ± 1.77, Bm2 = 51.09 ± 0.41 fmol/mg protein, n = 2 and K d1 = 0.61 ± 0.02, K d2 = 3.41 ± 0.13 nM, B m1 = 1.88 ± 0.028, B m2 = 9.27 ± 0.08 fmol/mg protein, n = 2, respectively. The concentration of active receptors (B max) increased twofold for both ligands. It was suggested that α1- and α2-adrenoceptors in rat cerebral cortex subcellular membrane fractions exist as dimers. A modulating influence of isoprenaline and propranolol on the specific binding of the antagonists to α1- and α2- adrenoceptors was revealed, which was manifested in the activating effect on the [3H]prazosin binding parameters, in the inhibitory effect on the [3H]RX821002 binding parameters, and in a change of the general character of binding for both ligands.

Hispidulin alleviated methamphetamine-induced hyperlocomotion by acting at α6 subunit-containing GABAA receptors in the cerebellum.

Hispidulin is a flavonoid we isolated from Clerodendrum inerme, an herb that effectively remitted a case of intractable motor tic disorders. Hispidulin was shown to be a positive allosteric modulator (PAM) of GABAA receptors, including the α6 subunit-containing subtype (α6GABAAR) that is predominantly expressed in cerebellar granule cells and insensitive to diazepam. We explored the action mechanism(s) of hispidulin using hyperdopaminergic mouse models induced by methamphetamine and apomorphine, based on the hyperdopaminergic nature of tic disorders. Hispidulin significantly inhibited methamphetamine-induced hyperlocomotion (MIH) at i.p. doses without affecting apomorphine-induced hyperlocomotion and stereotypy behaviors or having significant benzodiazepine-like effects (BZLE), including sedation, anxiety, and motor impairment. When given by intracerebellar (i.c.b.) microinjection, hispidulin also alleviated MIH and this effect was prevented by i.c.b. coadministration of furosemide, an α6GABAAR antagonist, and mimicked by i.c.b. Ro 15-4513, an α6GABAAR PAM. Conversely, i.c.b. diazepam did not affect MIH while it reduced MIH at i.p. doses having significant BZLE. In a screening assay for 92 neurotransmitter receptors/degradation enzymes/transporters, hispidulin displayed significant (>50% inhibition of radiolabeled ligand binding at 10μM) binding affinity only at the benzodiazepine binding site of GABAARs (IC50 0.73∼1.78μM) and catecholamine-o-methyl-transferase (COMT) (IC50 1.32μM). OR-486, a more potent COMT inhibitor than hispidulin, did not affect MIH. It is suggested that hispidulin alleviates MIH via acting as a PAM of cerebellar α6GABAARs, but not through COMT inhibition or affecting dopamine receptor responsiveness. Thus, selective α6GABAAR PAMs may have the potential to be a novel treatment for hyperdopaminergic disorders.

The peripheral cannabinoid receptor 1 antagonist VD60 efficiently inhibits carbon tetrachloride-intoxicated hepatic fibrosis progression

This study investigated a peripheral selective CB₁ antagonist 3,4,22-3-demethoxycarbonyl-3-hydroxylmethyl-4-deacetyl-vindoline 3,4-thionocarbonate (VD60) that efficiently inhibited hepatic fibrosis with lower psychological side effects. A competitive radiolabeled ligand binding experiment and 3'-5'-cyclic adenosine monophosphate (cAMP) response element-driven luciferase analysis were performed to evaluate the antagonistic activity of VD60. Cell viability and collagen production were examined in the human hepatic stellate cell (HSC) line LX-2 and primary cultured rat HSCs. The antifibrotic effects of VD60 were investigated in a CCl₄-induced liver fibrosis mouse model. The concentration of VD60 in the blood and the brain was determined by high-performance liquid chromatography-mass spectrum analysis. Furthermore, the potential underlying mechanisms of VD60 were investigated by Western blot. VD60 selectively competed with the radiolabeled CB1 agonist to bind to CB1. VD60 antagonized CB1 agonist-induced Akt phosphorylation and increased the accumulation of intracellular cAMP. VD60 strongly reduced the expression of α₂(I) pro-collagen mRNA and exerted potent antiproliferative effects on primary HSCs and LX-2 cells. The inhibition of reactive oxygen species production and phosphorylation of Akt, extracellular-signal-regulated kinase (ERK), and Smad3 may explain the underlying mechanisms behind the antiproliferative effect of VD60. Moreover, the in vivo antifibrotic activity of VD60 was confirmed in a CCl4-induced liver fibrosis mouse model. Most importantly, the concentration of VD60 in the peripheral blood was much higher than in the brain, suggesting that VD60 could act as a novel peripheral CB1 antagonist to efficiently inhibit hepatic fibrosis and could be used as a lead compound with low brain side effects in peripheral antifibrotic agents.

Paclitaxel Is an Inhibitor and Its Boron Dipyrromethene Derivative Is a Fluorescent Recognition Agent for Botulinum Neurotoxin Subtype A

We have successfully identified one new inhibitor and one new fluorescent recognition agent for the botulinum neurotoxin subtype A (BoNT/A) using the virtual screening protocol "protein scanning with virtual ligand screening" (PSVLS). Hit selection used an in-house developed holistic binding scoring method. Selected hits were tested experimentally for inhibitory activity using fluorescence resonance energy transfer (FRET) assays against the light chain (catalytic domain) of BoNT/A. Ligand binding was determined against the light and heavy chain BoNT/A complex through either radiolabeled ligand binding assays (nonfluorescent ligands) or fluorescence intensity assays (fluorescent ligands). These experimental assays have confirmed one compound (paclitaxel) to inhibit BoNT/A's proteolytic activity experimentally with an IC50 of 5.2 μM. A fluorescent derivative was also confirmed to bind to the toxin and therefore is a suitable candidate for the rational design of new detection agents and for the development of fluorescence-based multiprobe detection assays.

Honokiol blocks store operated calcium entry in CHO cells expressing the M3 muscarinic receptor: honokiol and muscarinic signaling

BackgroundHonokiol, a cell-permeable phenolic compound derived from the bark of magnolia trees and present in Asian herbal teas, has a unique array of pharmacological actions, including the inhibition of multiple autonomic responses. We determined the effects of honokiol on calcium signaling underlying transmission mediated by human M3 muscarinic receptors expressed in Chinese hamster ovary (CHO) cells. Receptor binding was determined in radiolabelled ligand binding assays; changes in intracellular calcium concentrations were determined using a fura-2 ratiometric imaging protocol; cytotoxicity was determined using a dye reduction assay.ResultsHonokiol had a potent (EC50 ≈ 5 μmol/l) inhibitory effect on store operated calcium entry (SOCE) that was induced by activation of the M3 receptors. This effect was specific, rapid and partially reversible, and was seen at concentrations not associated with cytotoxicity, inhibition of IP3 receptor-mediated calcium release, depletion of ER calcium stores, or disruption of M3 receptor binding.ConclusionsIt is likely that an inhibition of SOCE contributes to honokiol disruption of parasympathetic motor functions, as well as many of its beneficial pharmacological properties.

A comparison of in vitro methods for determining the membrane receptor expression in cell lines

Determining the number of expressed receptors per cell (NRPC) in cell lines is an important prerequisite for many experimental procedures in biomedical research. This paper focuses on the comparison of a newly developed method of determining NRPC - the Kinetic extrapolation method (KEX) - with the standard saturation method. These two methods, both based on radiolabeled ligand-receptor binding, were compared with the data on receptor expression found using quantified western blotting. Four cell lines with different expressions of epidermal growth factor receptor (EGFR) were chosen for the experiment: A431, HaCaT, HCT116 and HepG2. Two radiolabeled monoclonal antibodies specific for EGFR were used as ligands: [(131)I]-cetuximab and [(131)I]-panitumumab. The classic manual technique based on the saturation of cell receptors was performed on cells seeded in 24-well plates. The KEX method uses the LigandTracer, a special instrument which detects ligand retention in real time from seeded cells onto a rotating Petri dish. The western blot analysis was performed according to the routinely used procedure. A very close accordance between the manual saturation technique and the KEX method was found in all four cell lines used. The NRPC in the cell lines follows the same order using both ligands: A431>HaCaT>HCT116≈HepG2. Similarly, consistent data on EGFR expression in the studied cell lines were obtained using western blot analysis and the radiolabeled ligand binding assays. The KEX method could be as similarly useful for determining receptor expression as is the classic saturation method and western blotting.

P1-07-23: Absolute Quantification of Estrogen Receptor alpha in Breast Cancer.

Abstract Estrogen receptor alpha (ER) is the first biomarker to have been clinically validated as a predictor of cancer therapy response. Measurements of tumor ER expression were based on radiolabeled ligand binding to receptor present in tumor lysates. Despite substantial tissue requirement, lack of specificity for ER isoforms (alpha versus beta) or receptor integrity, quantitative determination of tumor ER content (fmol/mg total protein) was possible. Introduction of anti-ER antibodies later permitted immunohistochemical (***lHC) evaluation of ER expression. IHC determination of ER status in newly diagnosed breast cancer is now a standard of care. While these IHC assays have been shown to be as predictive of endocrine responsiveness as ligand binding assays, they remain semi-quantitative at best reporting tumor ER status either categorically (e.g. + or -) or as a numeric score which is subjective and lacks a linear relationship with endocrine responsiveness. Thus the lack of precision for quantifying ER as a predictive biomarker is one of the most important unresolved issues in breast cancer. We are working to develop a proteomic liquid chromatography-mass spectrometry (LC-MS) assay to help resolve this issue. Samples included recombinant ER (rER), immunoprecipitated (IP) rER, and IP ER from MCF7 cells. ER was digested with trypsin, lyophilised and solubilised in 5 femto-mol/microliter (100 μl) heavy peptide internal standard mix. ER peptides were resolved by LC (100 μl/minute) and detected by selected reaction monitoring MS. The area under the total ion chromatogram for each peptide were used to quantify the amount of analyte present in each sample as a single point reference to the signal of the heavy peptide spike. An 11 point calibration curve (0.1-1000 fmol on column (o/c)) of light peptides with each point in the curve spiked with 100 fmol heavy peptide was also produced to determine assay characteristics such as limits of detection (LOD), limits of quantification (LOQ), linearity, accuracy and precision. Three ER peptides were selected for quantification as they gave the greatest LOD, LOQ, linearity as well as reasonable intra- and inter-assay precision following multiple digestions of rER (intra = 3 digestions in 1 day; inter = 9 digestions over 3 weeks). Following IP of ER from four replicate MCF7 cell lysates (1mg/ml total protein) and measurement of ion intensities of the three ER peptides the mean concentration of ER was calculated to be 52 fmol (S.D of 7.5 fmol; n=4) per mg of total cell lysate after normalising for IP efficiency. We continue to develop the method to improve sensitivity and normalise for variability in IP and digestion. With the inclusion of reference peptides to known ER phosphorylation sites we are also in the process of quantifying ER phosphorylation. We aim to accurately determine ER concentration and phosphorylation status in tumor lysates and assess how these correlate with responsiveness to antiestrogen therapies. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-23.

The C-terminal segment of the second extracellular loop of the thromboxane A 2 receptor plays an important role in platelet aggregation

There is considerable interest in discovering novel antiplatelet approaches with an enhanced safety profile. To this end, in our efforts to define new targets for antithrombotic activity, we investigated the utility of antibodies which recognize the ligand binding domains of the platelet thromboxane A 2 receptor (TPR). We hypothesized that an antibody (abbreviated as C-EL2Ab), which interacts with the C-terminus of the second extracellular loop (C-EL2; i.e., ligand binding domain) of TPR exhibits antagonistic activity. Our findings demonstrate that C-EL2Ab did indeed inhibit TPR-mediated platelet aggregation. However, it was devoid of any apparent effects on aggregation triggered by ADP or the thrombin receptor activating peptides 1 or 4. Furthermore, results from radiolabeled ligand binding studies indicate that C-EL2Ab competitively displaced the classical TPR antagonist [ 3H]SQ29,548 from its binding sites. On the other hand, control experiments indicated that normal rabbit IgG and an antibody which targets a TPR domain separate from those involved in ligand recognition, failed to inhibit aggregation in response to TPR activation. Collectively, these findings demonstrate that C-EL2 of TPR plays a critical role in platelet activation, and establish C-EL2Ab as a function blocking antibody. Furthermore, our data suggest a potential for the therapeutic application of C-EL2Ab, which may serve either as an alternative to, or a complement for current treatments. Finally, the identification of a functionally active TPR sequence should aid molecular modeling study predictions for organic derivatives which possess in vivo activity.

P38 Mitogen–activated Protein Kinase Activation by Nerve Growth Factor in Primary Sensory Neurons Upregulates μ-Opioid Receptors to Enhance Opioid Responsiveness Toward Better Pain Control

Sensory neuron opioid receptors are targets for spinal, epidural, and peripheral opioid application. Although local nerve growth factor (NGF) has been identified as a mediator of sensory neuron μ-opioid receptor (MOR) up-regulation, the signaling pathways involved have not been yet identified. Wistar rats were treated with intraplantar vehicle, Freund's complete adjuvant, NGF, NGF plus intrathecal p38 mitogen-activated protein kinase (MAPK) inhibitors, or NGF plus extracellular signal-regulated kinase-1/2 MAPK inhibitors. After 4 days of treatment, paw pressure thresholds of an intraplantar full (fentanyl) or partial (buprenorphine) opioid agonist were determined by algesiometry. Tissue samples from rat dorsal root ganglia were subjected to radiolabeled ligand binding, Western blot analysis, and confocal immunofluorescence. Exogenous and endogenous NGF resulting from Freund's complete adjuvant inflammation produced significant potentiation and enhanced efficacy in fentanyl- and buprenorphine-induced dose-dependent antinociception, respectively. Furthermore, in the ipsilateral dorsal root ganglia, NGF produced a significant increase in MOR binding sites, proteins, and immunoreactive neurons. In parallel, phosphorylated p38-MAPK protein, the number of phosphorylated p38-MAPK immunoreactive neurons expressing MOR in dorsal root ganglia, and the peripherally directed axonal transport of MOR significantly increased. Finally, NGF-induced effects occurring in dorsal root ganglia, on axonal transport, and on the potentiation or enhanced efficacy of opioid antinociception were abrogated by inhibition of p38, but not extracellular signal-regulated kinase-1/2, MAPK. Local NGF through activation of the p38-MAPK pathway leads to adaptive changes in sensory neuron MOR toward enhanced susceptibility to local opioids. This effect may act as a counter-regulatory response to p38-MAPK-induced pain (e.g., inflammatory pain) to facilitate opioid-mediated antinociception.

Gi protein coupling to adenosine A1–A2A receptor heteromers in human brain caudate nucleus

Pharmacological characterization of adenosine A(1) and A(2A) receptors in human brain caudate nucleus membranes led to non-cooperative binding of radiolabelled ligands. In human caudate nucleus but not in cortex, the agonist binding to A(1) receptors was modulated by the agonist binding to A(2A) receptors indicating a functional negative cross-talk. Accordingly, the A(1) receptor-activation-mediated G(i)-dependent guanosine 5'-o-(3-[(35)S]thio-triphosphate) binding was modulated by agonist binding to A(2A) receptors. A(2A) receptors occupation led to a decrease in the potency of A(1) receptor agonists. These results indicate that A(1) but not A(2A) receptors activation, likely occurring at low adenosine concentrations, engages a G(i)-mediated signaling; however, when both receptors are occupied by adenosine, there is an A(2A) receptor-mediated impairment of G(i)-operated transducing units. These findings are relevant to get insight into the complex relationships derived from co-expression of multiple neurotransmitter/neuromodulator receptors subtypes that individually are coupled to different G proteins. A further finding was the demonstration that the A(2A) receptor agonist, CGS 21680, at high concentrations able to significantly bind to the A(1) receptor, behaved as a partial agonist of the later receptor. This fact might be taken into account when characterizing CGS 21680 actions in human cells expressing A(1) receptors when the compound is used at micromolar concentrations.

CD69 Suppresses Sphingosine 1-Phosophate Receptor-1 (S1P1) Function through Interaction with Membrane Helix 4

Lymphocyte egress from lymph nodes requires the G-protein-coupled sphingosine 1-phosphate receptor-1 (S1P1). The activation antigen CD69 associates with and inhibits the function of S1P1, inhibiting egress. Here we undertook biochemical characterization of the requirements for S1P1-CD69 complex formation. Domain swapping experiments between CD69 and the related type II transmembrane protein, NKRp1A, identified a requirement for the transmembrane and membrane proximal domains for specific interaction. Mutagenesis of S1P1 showed a lack of requirement for N-linked glycosylation, tyrosine sulfation, or desensitization motifs but identified a requirement for transmembrane helix 4. Expression of CD69 led to a reduction of S1P1 in cell lysates, likely reflecting degradation. Unexpectedly, the S1P1-CD69 complex exhibited a much longer half-life for binding of S1P than S1P1 alone. In contrast to wild-type CD69, a non-S1P1 binding mutant of CD69 failed to inhibit T cell egress from lymph nodes. These findings identify an integral membrane interaction between CD69 and S1P1 and suggest that CD69 induces an S1P1 conformation that shares some properties of the ligand-bound state, thereby facilitating S1P1 internalization and degradation.

Abstract 5532: Folate receptor expression in human malignancies to select patients who would benefit from folate conjugated therapy

Abstract The folate receptor (FR) is expressed on a number of human tumors, including ovarian, renal, lung, and breast. Folic acid is the ligand for the FR and many innovative strategies for targeting therapeutics to tumors make use of this ligand/receptor pair. Novel therapeutic approaches include folate conjugation to cytotoxics or radiopharmaceuticals, folate-coated liposomes containing antisense oligonucleotides, folate conjugates of pro-drugs, and folate-linked nanoparticle carriers for therapeutic drugs and genes. Potential patient enrichment strategies are necessary to allow for better targeting of these agents to those patients who have FR expressing tumors. Preclinical experiments to evaluate the molecular correlation of FR levels by three different methods, qRT-PCR, IHC, and the radiolabeled ligand binding assay, were conducted. A total of seventeen samples (four xenografts and thirteen primary patient ovarian and endometrial tumor biopsies) were evaluated for FR expression in these three independent assays and the FR levels correlated in this sample size. Compared to a preclinical model that was sensitive to epothilone-folate conjugate therapy, most patient samples expressed higher FR levels. These data provide a reliable and useful method to identify patient tumors that express the FR above a baseline determined to be sufficient for response to folate-conjugated therapy in preclinical models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5532.

The specific binding sites of eyestalk- and pericardial organ-crustacean hyperglycaemic hormones (CHHs) in multiple tissues of the blue crab,Callinectes sapidus

Crustacean hyperglycaemic hormone from the pericardial organ (PO-CHH) is a CHH-related neuropeptide but its function and target tissues are not known in crustaceans. To investigate this issue, we employed radiolabelled ligand binding and cGMP assays, using eyestalk-CHH (ES-CHH) as a reference neuropeptide. The membranes were prepared from various tissues of Callinectes sapidus: hepatopancreas, hindgut, midgut, gills, heart, abdominal muscles and scaphognathites. Like ES-CHH, recombinant PO-CHH (rPO-CHH) specifically bound to the membranes of scaphognathites=abdominal muscles>midgut>gills> heart>hindgut and hepatopancreas (list order corresponds to the number of binding sites). The specific binding sites of (125)I-ES-CHH in hepatopancreas and gills were saturable and displaceable. The abdominal muscle membrane binding sites were specific and saturable to both CHHs. These binding sites were displaced by homologous neuropeptides, but poorly displaced by the heterologous counterpart. As for the second messenger, the expected increment (3- to >20-fold) in the amount of cGMP produced by ES-CHH was noted in most tissues tested except midgut. Recombinant PO-CHH increased cGMP production 1.5- to 4-fold in scaphognathites, heart, midgut, hindgut and abdominal muscles. The results obtained from the binding study suggest that PO-CHH also has multiple target tissues of which abdominal muscles and scaphognathites are the primary ones. The differences in the primary amino acid sequences of PO-CHH and ES-CHH, particularly in the C-terminal region and in the amidation at C-terminus, may contribute to the truncated responses of hyperglycaemia, cGMP stimulation and binding affinity.

Potent in vitro and in vivo antitumor activity of interleukin‐4‐conjugated Pseudomonas exotoxin against human biliary tract carcinoma

Targeting cytotoxins or immunotoxins to tumor cell surface receptors represents a new approach for the treatment of cancers. We tested the antitumor activity of a cytotoxin (IL-4-PE) composed of an interleukin-4 (IL-4) targeting moiety and a truncated form of Pseudomonas exotoxin A against human biliary tract carcinoma (BTC). Immunohistochemical analysis showed that cultured BTC cell lines and cancerous epithelia in BTC tissue (e.g., gallbladder carcinoma, extrahepatic cholangiocarcinoma, and intrahepatic cholangiocarcinoma) expressed receptors for IL-4 in situ at high densities. However, normal epithelial cells in gallbladder and bile duct tissues did not express these IL-4 receptors. Eight BTC cell lines expressed IL-4R on the cell surface as determined by radiolabeled ligand binding assays. When these cells were treated with IL-4-PE, significant cytotoxicity was observed as determined by the inhibition of protein synthesis. The concentration of agent causing 50% inhibition of protein synthesis (IC(50)) was found to be less than 10 ng/mL in 4 of the 8 BTC cell lines studied. The antitumor activity of IL-4-PE was assessed for human BTC cells implanted subcutaneously in immunodeficient mice. By intratumoral injection of IL-4-PE, complete disappearance of the established tumors was observed in 40% of animals. Intraperitoneal administration of IL-4-PE at tolerated doses to animals with peritoneally disseminated BTC exhibited significantly prolonged survival compared to untreated animals (>14 weeks vs. 5 weeks in treated and untreated mice, respectively). These results indicate that IL-4 receptor-targeted cytotoxin is a potent agent that may provide a new therapeutic option for BTC.

L-DOPA Is an Endogenous Ligand for OA1

Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE). How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1) expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR) activation, including influx of intracellular calcium and recruitment of β-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation.