Measurement Of Ligand Binding Research Articles

Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity

The Gibbs free energy difference between native and unfolded states ("stability") is one of the fundamental characteristics of a protein. By exploiting the thermodynamic linkage between ligand binding and stability, interactions of a protein with small molecules, nucleic acids, or other proteins can be detected and quantified. Determination of protein stability can therefore provide a universal monitor of biochemical function. Yet, the use of stability measurements as a functional probe is underutilized, because such experiments traditionally require large amounts of protein and special instrumentation. Here we present the quantitative cysteine reactivity (QCR) technique to determine protein stabilities rapidly and accurately using only picomole quantities of material and readily accessible laboratory equipment. We demonstrate that QCR-derived stabilities can be used to measure ligand binding over a wide range of ligand concentrations and affinities. We anticipate that this technique will have broad applications in high-throughput protein engineering experiments and functional genomics.

Identification, characterization and genetic mapping of TLR1 loci in rainbow trout ( Oncorhynchus mykiss)

Induction of innate immune pathways is critical for early anti-microbial defense but there is limited understanding of how teleosts recognize microbial molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 1 and 2 form a heterodimer involved in recognizing peptidoglycans and lipoproteins of microbial origin. Herein, we identify and describe the rainbow trout ( Oncorhynchus mykiss) TLR1 gene ortholog and its mRNA expression. Two TLR1 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA sequencing and genetic linkage analyses. Full length cDNA clone and direct sequencing of four BACs revealed an intact omTLR1 open reading frame (ORF) located on chromosome 14 and a second locus on chromosome 25 that contains a TLR1 pseudogene. The duplicated trout loci exhibit conserved synteny with other fish genomes that extends beyond the TLR1 gene sequences. The omTLR1 gene includes a single large coding exon similar to all other described TLR1 genes, but unlike other teleosts it also has a 5′ UTR exon and intron preceding the large coding exon. The omTLR1 ORF is predicted to encode an 808 amino-acid protein with 69% similarity to the Fugu TLR1 and a conserved pattern of predicted leucine-rich repeats (LRR). Phylogenetic analysis grouped omTLR1 with other fish TLR1 genes on a separate branch from the avian TLR1 and mammalian TLR1, 6 and 10. omTLR1 expression levels in rainbow trout anterior kidney leukocytes were not affected by the human TLR2/ 6 and TLR2/ 1 agonists diacylated lipoprotein (Pam 2CSK 4) and triacylated lipoprotein (Pam 3CSK 4). However, due to the lack of TLR6 and 10 genes in teleost genomes and up-regulation of TLR1 mRNA in response to LPS and bacterial infection in other fish species we hypothesize an important role for omTLR1 in anti-microbial immunity. Therefore, the identification of a TLR2 ortholog in rainbow trout and the development of assays to measure ligand binding and downstream signaling are critical for future elucidation of omTLR1 functions.

Voltage Gated Trapping of fcAMP in HCN2 Channels

HCN channels are nonselective tetrameric cation channels that are activated by hyperpolarizing voltages and modulated by the ligand cAMP. They generate spontaneous rhythmic activity in the heart and brain. Ligand binding to the intracellular cyclic nucleotide-binding site accelerates the activation kinetics, shifts the steady-state activation to more positive voltages and increases the open probability. We expressed homotetrameric HCN2 channels in Xenopus oocytes. Using fast solution exchange by a piezoelectric system we observed that removal of cAMP from the patch induces significant current deactivation if the channels are weakly activated by voltage but only minor deactivation if the channels are strongly activated by voltage. To visualize ligand unbinding directly, simultaneous measurement of activation and ligand binding was performed in excised inside-out macropatches by means of patch-clamp fluorometry with confocal resolution, using a fluorescent cAMP derivate (fcAMP) that activates the channels closely similar to cAMP. As a result, fast removal of saturating fcAMP (7.5 μM) from maximally activated HCN2 channels (−130 mV) caused a fluorescence decay with a fast and a slow component. Assuming four bound ligands per channel at a saturating ligand concentration and at saturating hyperpolarization, the amplitudes of the two exponentials of ∼50% each suggest that two of the for bound ligands unbind fast, whereas the remaining two are trapped. This trapping can be shown to depend on channel activation. Comparing the time courses of deactivation and unbinding following a fast removal of the ligand, the results make it likely that only two of the four binding sites have to be occupied to cause maximal ligand induced modulation, resembling the situation in related CNGA2 channels, where two of the four ligands are sufficient to open the channel maximally.

Pharmacological Characterization of Receptor Redistribution and β-Arrestin Recruitment Assays for the Cannabinoid Receptor 1

Receptor redistribution and beta-arrestin recruitment assays provide a G-protein-subtype-independent method to measure ligand-stimulated activation of G-protein-coupled receptors. In particular beta-arrestin assays are becoming an increasingly popular tool for drug discovery. The authors have compared a high-content-imaging-based Redistribution assay and 2 nonimaging-based beta-arrestin recruitment assays, Tango and PathHunter, for the cannabinoid receptor 1. Inasmuch as all 3 assays use receptors that are modified at the C-terminus, the authors verified their pharmacology via detection of Galpha(i) coupling of the receptor in cAMP assays using reference ligands. The potencies and efficacies of the cannabinoid receptor agonists CP55,940 and WIN55,212-2 correlated well between the 3 assays, and are comparable with the measured ligand binding affinities. The inverse agonist SR141716 decreased basal signal in all 3 assays, but only in the Tango bla assay a reliable EC50 could be determined for this compound, suggesting that Tango is the most suitable assay for the identification of new inverse agonists. Both the Redistribution and the PathHunter assay could discriminate partial agonists from full agonists, whereas in the Tango assay partial agonists behaved as full agonists. Only the PathHunter cells allowed detection of cannabinoid receptor activation via beta-arrestin recruitment and Galpha(i)-protein-mediated inhibition of cAMP, thus enabling the identification of biased ligands that differ in these cellular effects. The characteristics and limitations of the different assays are discussed.

Ligand-binding by catalytically inactive mutants of the cblB complementation group defective in human ATP:cob(I)alamin adenosyltransferase

MMAB (methylmalonic aciduria type B) is a mitochondrial enzyme involved in the metabolism of vitamin B 12. It functions as the ATP:cob(I)alamin adenosyltransferase for the generation of adenosylcobalamin (AdoCbl), the cofactor of methylmalonyl-CoA mutase (MCM). Impaired MMAB activity leads to the inherited disorder methylmalonic aciduria and is responsible for the cblB complementation group. In this study, the effects on substrate binding of two catalytically inactive patient mutations, R190H and R186W, were investigated using intrinsic fluorescence quenching of MMAB as a measure of ligand-binding. We report the dissociation constant ( K d) of wild-type MMAB for HOCbl is 51 μM and for ATP is 365 μM and show that cobalamin enhances the affinity of MMAB for ATP, while ATP does not show detectable effects on cobalamin binding. We confirm that residue Arg190 plays a role in the formation of the ATP-binding site as described previously [H.L. Schubert, C.P. Hill, Structure of ATP-bound human ATP:cobalamin adenosyltransferase, Biochemistry 45 (2006) 15188–15196]. Unexpectedly, mutation R186W does not disrupt the binding of HOCbl to MMAB as predicted; instead, both R190H and R186W significantly disrupt the affinity between MMAB and AdoCbl. We surmise that these two residues may be critical for the transfer of the 5′-deoxyadenosyl group from ATP to cob(I)alamin, possibly by contributing to the precise positioning of the two substrates to permit catalysis to occur. Characterization of ligand-binding by MMAB provides insight into the mechanism of cobalamin adenosylation and the effect of patient mutations in the inherited disorder.

Regional changes in the cholinergic system in mice lacking monoamine oxidase A

Elevated brain monoamine concentrations resulting from monoamine oxidase A genetic ablation (MAOA knock-out mice) lead to changes in other neurotransmitter systems. To investigate the consequences of MAOA deficiency on the cholinergic system, we measured ligand binding to the high-affinity choline transporter (CHT1) and to muscarinic and nicotinic receptors in brain sections of MAOA knock-out (KO) and wild-type mice. A twofold increase in [ 3H]-hemicholinium-3 ([ 3H]-HC-3) binding to CHT1 was observed in the caudate putamen, nucleus accumbens, and motor cortex in MAOA KO mice as compared with wild-type (WT) mice. There was no difference in [ 3H]-HC-3 labeling in the hippocampus (dentate gyrus) between the two genotypes. Binding of [ 125I]-epibatidine ([ 125I]-Epi), [ 125I]-α-bungarotoxin ([ 125I]-BGT), [ 3H]-pirenzepine ([ 3H]-PZR), and [ 3H]-AFDX-384 ([ 3H]-AFX), which respectively label high- and low-affinity nicotinic receptors, M1 and M2 muscarinic cholinergic receptors, was not modified in the caudate putamen, nucleus accumbens, and motor cortex. A small but significant decrease of 19% in M1 binding densities was observed in the hippocampus (CA1 field) of KO mice. Next, we tested acetylcholinesterase activity and found that it was decreased by 25% in the striatum of KO mice as compared with WT mice. Our data suggest that genetic deficiency in MAOA enzyme is associated with changes in cholinergic activity, which may account for some of the behavioral alterations observed in mice and humans lacking MAOA.

His-311 and Arg-559 Are Key Residues Involved in Fatty Acid Oxygenation in Pathogen-inducible Oxygenase

Pathogen-inducible oxygenase (PIOX) oxygenates fatty acids into 2R-hydroperoxides. PIOX belongs to the fatty acid alpha-dioxygenase family, which exhibits homology to cyclooxygenase enzymes (COX-1 and COX-2). Although these enzymes share common catalytic features, including the use of a tyrosine radical during catalysis, little is known about other residues involved in the dioxygenase reaction of PIOX. We generated a model of linoleic acid (LA) bound to PIOX based on computational sequence alignment and secondary structure predictions with COX-1 and experimental observations that governed the placement of carbon-2 of LA below the catalytic Tyr-379. Examination of the model identified His-311, Arg-558, and Arg-559 as potential molecular determinants of the dioxygenase reaction. Substitutions at His-311 and Arg-559 resulted in mutant constructs that retained virtually no oxygenase activity, whereas substitutions of Arg-558 caused only moderate decreases in activity. Arg-559 mutant constructs exhibited increases of greater than 140-fold in Km, whereas no substantial change in Km was observed for His-311 or Arg-558 mutant constructs. Thermal shift assays used to measure ligand binding affinity show that the binding of LA is significantly reduced in a Y379F/R559A mutant construct compared with that observed for Y379F/R558A construct. Although Oryza sativa PIOX exhibited oxygenase activity against a variety of 14-20-carbon fatty acids, the enzyme did not oxygenate substrates containing modifications at the carboxylate, carbon-1, or carbon-2. Taken together, these data suggest that Arg-559 is required for high affinity binding of substrates to PIOX, whereas His-311 is involved in optimally aligning carbon-2 below Tyr-379 for catalysis.

Meta‐analysis of cannabinoid ligand binding affinity and receptor distribution: interspecies differences

A meta-analysis, unlike a literature review, synthesizes previous studies into new results. Pooled data from 211 studies measured ligand binding affinities at human (Hs) or rat (Rn) cannabinoid receptors CB1 and CB2. Cochrane methods were modified for this non-clinical analysis. Meta-regression detected data heterogeneity arising from methodological factors: use of sectioned tissues, lack of PMSF and choice of radioligand. Native brain tissues exhibited greater affinity (lower nM) than transfected cells, but the trend fell short of significance, as did the trend between centrifugation and filtration methods. Correcting for heterogeneity, mean Ki values for delta 9-tetrahydrocannabinol differed significantly between HsCB1 and RnCB1 (25.1 and 42.6 nM, respectively) but not between HsCB1 and HsCB2 (25.1 and 35.2). Mean Kd values for HsCB1, RnCB1 and HsCB2 of CP55,940 (2.5, 0.98, 0.92) and WIN55,212-2 (16.7, 2.4, 3.7) differed between HsCB1 and RnCB1 and between HsCB1 and HsCB2. SR141716A differed between HsCB1 and RnCB1 (2.9 and 1.0 nM). Anandamide at HsCB1, RnCB1 and HsCB2 (239.2, 87.7, 439.5) fell short of statistical differences due to heterogeneity. We consider these Kd and Ki values to be the most valid estimates in the literature. Sensitivity analyses did not support the numerical validity of cannabidiol, cannabinol, 2-arachidonoyl glycerol and all ligands at RnCB2. Aggregate rank order analysis of CB(1) distribution in the brain (pooled from 119 autoradiographic, immunohistochemical and in situ hybridization studies) showed denser HsCB1 expression in cognitive regions (cerebral cortex) compared to RnCB1, which was relatively richer in movement-associated areas (cerebellum, caudate-putamen). Implications of interspecies differences are discussed.

Temperature-dependent heme kinetics with nonexponential binding and barrier relaxation in the absence of protein conformational substates

We present temperature-dependent kinetic measurements of ultrafast diatomic ligand binding to the "bare" protoheme (L(1)-FePPIX-L(2), where L(1) = H(2)O or 2-methyl imidazole and L(2) = CO or NO). We found that the binding of CO is temperature-dependent and nonexponential over many decades in time, whereas the binding of NO is exponential and temperature-independent. The nonexponential nature of CO binding to protoheme, as well as its relaxation above the solvent glass transition, mimics the kinetics of CO binding to myoglobin (Mb) but on faster time scales. This demonstrates that the nonexponential kinetic response observed for Mb is not necessarily due to the presence of protein conformational substates but rather is an inherent property of the solvated heme. The nonexponential kinetic data were analyzed by using a linear coupling model with a distribution of enthalpic barriers that fluctuate on slower time scales than the heme-CO recombination time. Below the solvent glass transition (T(g) approximately 180 K), the average enthalpic rebinding barrier for H(2)O-PPIX-CO was found to be approximately 1 kJ/mol. Above T(g), the barrier relaxes and is approximately 6 kJ/mol at 290 K. Values for the first two moments of the heme doming coordinate distribution extracted from the kinetic data suggest significant anharmonicity above T(g). In contrast to Mb, the protoheme shows no indication of the presence of "distal" enthalpic barriers. Moreover, the wide range of Arrhenius prefactors (10(9) to 10(11) s(-1)) observed for CO binding to heme under differing conditions suggests that entropic barriers may be an important source of control in this class of biochemical reactions.

A platelet biomarker for assessing phosphoinositide 3-kinase inhibition during cancer chemotherapy

Thrombin cleavages of selective proteinase-activated receptors (PAR) as well as PAR-activating peptide ligands can initiate the phosphoinositide 3-kinase (PI3K) signaling cascade in platelets. Downstream to this event, fibrinogen receptors on platelets undergo conformational changes that enhance fibrinogen binding. In our study, we used this phenomenon as a surrogate biomarker for assessing effects on PI3K activity. Our method, using flow cytometric measurement of fluorescent ligand and antibody binding, uncovered a 16- to 45-fold signal window after PAR-induced platelet activation. Pretreatment (in vitro) with the PI3K inhibitors wortmannin and LY294002 resulted in concentration-dependent inhibition at predicted potencies. In addition, platelets taken from mice treated with wortmannin were blocked from PAR-induced ex vivo activation concomitantly with a decrease in phosphorylation of AKT from excised tumor xenografts. This surrogate biomarker assay was successfully tested (in vitro) on blood specimens received from volunteer cancer patients. Our results indicate that measurement of platelet activation could serve as an effective drug activity biomarker during clinical evaluation of putative PI3K inhibitors.

Real-Time Measurement of Myosin–Nucleotide Noncovalent Complexes by Electrospray Ionization Mass Spectrometry

Nanoelectrospray ionization mass spectrometry has been used to measure the binding of ATP and ADP to the active site of rabbit skeletal myosin-S1. Increases in the molecular mass of myosin-S1 of 425 ± 10 Da were obtained with the binding of ADP to the active site and by 530 ± 10 Da with either ATP or hydrolysis products ADP and phosphate. Active site titrations of myosin-S1 with ADP gave a stoichiometry of ∼1 ADP/S1 with an affinity in the micromolar range. The binding of ATP to myosin-S1 could be observed in the presence of up to 60 μM of excess MgATP without nonspecific binding of MgATP to the myosin. Conversion of the nucleotide complex containing an equilibrium mixture of ATP and ADP-Pi bound to myosin-S1 to one containing only bound ADP occurs at a rate consistent with that of the known steady-state rate of ATP hydrolysis. We expect this method to be of considerable use in the analysis of ligand binding and hydrolysis by the active sites of expressed myosin and myosin subfragments, which are not available in sufficient quantities for conventional methods of measurement of ligand binding.

Immobilizing BK-channels in artificial lipid bilayers using annexin V

In this report, we show an improved method for the simultaneous measurement of optical and electrical properties of single-channel proteins for analysis of the gating mechanism. Large-conductance Ca2+-activated potassium (BK) channels were isolated from porcine uterine smooth muscle and labeled with Cy5 via antibody against the N-terminal. These Cy5-labeled BK channels were incorporated into lipid bilayer membranes followed by single channel current measurements. Cy5-labeled BK channels possessed Ca2+ and voltage sensitivity. The orientation of the vesicles was determined to be outside-out. Charybdotoxin applied from the cis side blocked the channel current. For stable observations of ligand and channel binding, BK channel immobilization was also examined. The lateral diffusion coefficient of BK channels decreased over 200 fold in 1 μ annexin V while the open probability did not change. This study is a significant advancement in simultaneous measurements of ligand binding and current change at the single channel level. [DOI: 10.1380/ejssnt.2007.1]

Sordarin Derivatives Induce a Novel Conformation of the Yeast Ribosome Translocation Factor eEF2

The sordarins are fungal specific inhibitors of the translation factor eEF2, which catalyzes the translocation of tRNA and mRNA after peptide bond formation. We have determined the crystal structures of eEF2 in complex with two novel sordarin derivatives. In both structures, the three domains of eEF2 that form the ligand-binding pocket are oriented in a different manner relative to the rest of eEF2 compared with our previous structure of eEF2 in complex with the parent natural product sordarin. Yeast eEF2 is also shown to bind adenylic nucleotides, which can be displaced by sordarin, suggesting that ADP or ATP also bind to the three C-terminal domains of eEF2. Fusidic acid is a universal inhibitor of translation that targets EF-G or eEF2 and is widely used as an antibiotic against Gram-positive bacteria. Based on mutations conferring resistance to fusidic acid, cryo-EM reconstructions, and x-ray structures of eEF2, EF-G, and an EF-G homolog, we suggest that the conformation of EF-G stalled on the 70 S ribosome by fusidic acid is similar to that of eEF2 trapped on the 80 S ribosome by sordarin.

The Evolution of GABAA Receptor–Like Genes

The inhibitory ligand-gated ion channel family of receptors, including the type A gamma-aminobutryic acid (GABA(A)) and glycine receptors, mediates inhibitory neurotransmissions in the central nervous system. In this study, GABA receptor (GABR) evolution was explored through comparative genomics using genomes that span divergent lineages. GABA(A)/Gly receptor-like (GRL) gene sequences were retrieved from the genomes of various species ranging from mammal to fish to worm and subjected to cross-species comparison. All vertebrate GRL gene sets in the study but no invertebrate ones exhibit the extensive and conserved pattern of gene clustering that is characteristic of human GABR genes, indicating that the gene clusters were established early in vertebrate evolution, after divergence from the invertebrates. Moreover, the vertebrate gene structure is highly conserved with a basic 9-coding exon structure, whereas, as well as being diverse in copy numbers and chromosomal loci, the invertebrate GRL genes display a variety of gene structures. Remarkably, the invertebrates each possess a unique GRL gene pair that lies in neighboring loci within their respective genomes: zc482.5 and zc482.1 in roundworm, CG8916 and CG17336 in fruitfly, Ci4249 and Ci4254 in Ciona, and these were revealed by phylogenetic analysis to be homologous to human GABR alpha and beta subunits, respectively. The phylogenetic classification of these genes is also corroborated by experimental ligand-binding measurements using recombinant gene products. Furthermore, the 3 invertebrate gene pairs harbor characteristic key residues and exhibit similarities in intron positions to their vertebrate counterparts. The results strongly indicate that such a gene pair originally existed in the bilaterian ancestor from which all 3 phyla evolved and suggest that the extant GABR clusters arose from an ancestral alpha-beta subunit gene pair gave rise to the extant GABR clusters.

Expression and purification of phosphorylated and non-phosphorylated human MEK1

Kinases exist in either a high or low activity form depending on the phosphorylation state of the activating lip. These two different forms of the same kinase may adopt different conformations that affect not only activity but also inhibitor binding and the ability to crystallize the protein. Therefore, isolation of homogenous preparations of the phosphorylated and non-phosphorylated versions of a kinase is critical for accurate biophysical measurements of activity, stability and ligand binding as well as for protein crystallization. The aim of the present study is the expression, purification and characterization of recombinant human MEK1 protein in both the activated and low-activity states. A baculovirus co-expression system was developed for obtaining high levels of activated, phosphorylated human MEK1 kinase. High-Five cells were co-infected with human MEK1 virus and Raf-BXB, an untagged constitutively active version of Raf which is the activating kinase for MEK1. Unphosphorylated MEK1 was generated by treating MEK1 isolated from High-Five baculovirus expression with λ-phosphatase. The proteins were characterized by SDS–PAGE, LC–MS, Western blotting, enzymatic activity, and circular dichroism. Previous reports of MEK1 expression and purification yielded lower levels of protein and purity. The yield using High-Five cells was 5 mg/L for phosphorylated MEK1 and 10 mg/L for unphosphorylated MEK1. For phosphorylated MEK1, the specific activity was 3530 U/mg, the IC 50 values for the non-specific kinase inhibitors K252a and K252b were 8 and 47 nM, respectively, and the IC 50 for the MEK1 non-ATP competitive inhibitor, PD0325901, was 43 nM.

Fluorescent leukotriene B4: potential applications

Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation that acts primarily via a seven-transmembrane-spanning, G-protein-coupled receptor denoted BLT1. Here, we describe the synthesis and characterization of fluorescent analogs of LTB4 that are easy to produce, inexpensive, and without the disadvantages of a radioligand. Fluorescent LTB4 is useful for labeling LTB4 receptors for which no antibodies are available and for performing one-step fluorescence polarization assays conducive to high-throughput screening. We found that orange and green fluorescent LTB4 were full agonists that activated the LTB4 receptor BLT1 with EC50 values of 68 and 40 nM, respectively (4.5 nM for unmodified LTB4). Flow cytometric measurements and confocal imaging showed that fluorescent LTB4 colocalized with BLT1. Fluorescence polarization measurements showed that orange fluorescent LTB4 bound to BLT1 with a Kd of 66 nM and that this binding could be displaced by unlabeled LTB4 and other BLT1-specific ligands. Fluorescent LTB4 analogs were also able to displace tritiated LTB4. Orange fluorescent LTB4 binding to enhanced green fluorescent protein-tagged BLT1 could be observed using fluorescence resonance energy transfer. In addition to being a useful alternative to radiolabeled LTB4, the unique properties of fluorescently labeled LTB4 allow a variety of detection technologies to be used.

Global Kinase Screening. Applications of Frontal Affinity Chromatography Coupled to Mass Spectrometry in Drug Discovery

Utilizing frontal affinity chromatography with mass spectrometry detection (FAC-MS), we have identified novel applications in the discovery of small-molecule hits to protein targets that are difficult if not impossible to accomplish using traditional assays. We demonstrate for the first time an ability to distinguish between competitive ligands for the ATP and substrate sites of protein kinase C independently in the same experiment and show that ATP competitive ligands using a functionally inactive receptor tyrosine kinase can be identified. This ability of FAC-MS to simultaneously monitor binding at the ATP and substrate binding sites, as well as measure ligand binding to both active and inactive kinases, suggests that FAC-MS can be used as a "global kinase binding assay".

Low Levels of Estrogen Receptor β Protein Predict Resistance to Tamoxifen Therapy in Breast Cancer

Breast cancer is a hormone-dependent cancer, and the presence of estrogen receptor alpha (ER-alpha) in tumors is used clinically to predict the likelihood of response to hormonal therapies. The clinical value of the second recently identified ER isoform, called ER-beta, is less clear, and there is currently conflicting data concerning its potential role as a prognostic or predictive factor. To assess whether ER-beta expression is associated with clinical outcome, protein levels were measured by immunoblot analysis of a retrospective bank of tumor cell lysates from 305 axillary node-positive patients. A total of 119 received no adjuvant therapy, and 186 were treated with tamoxifen only. The median follow-up time was 65 months. Univariate and multivariate Cox regression modeling was done to assess the prognostic and predictive significance of ER-beta expression. Expression of ER-beta protein did not correlate significantly with any other clinical variables, including ER and progesterone levels (as measured ligand binding assay), tumor size, age, or axillary nodal status. In the untreated population, those patients whose tumors who expressed both receptor isoforms exhibited the most favorable outcome as compared with those patients who had lost ER-alpha expression. However, there was no association between ER-beta levels alone and either disease-free or overall survival in the untreated patient population. In contrast, in both univariate and multivariate analyses, high levels of ER-beta predicted an improved disease-free and overall survival in patients treated with adjuvant tamoxifen therapy. These findings provide evidence that ER-beta may be an independent predictor of response to tamoxifen in breast cancer. Furthermore, these results suggest that ER-beta may influence tumor progression in ways different from those mediated by the ER-alpha isoform.

Characterization of Melanocortin Receptors

This unit describes a Scintillation Proximity Assay (SPA) for the measurement of ligand binding to melanocortin receptors (MCRs) using membranes prepared from cell lines stably expressing recombinant MCRs. It provides a facile method for determining the affinity of compounds at MC1R, MC3R, MC4R, or MC5R.

Increased ventral striatal monoaminergic innervation in Tourette syndrome

Excessive striatal dopaminergic innervation is suggested to underlie Tourette syndrome (TS). Prior imaging and postmortem studies yield conflicting data. The authors used PET with the type 2 vesicular monoamine transporter ligand [(11)C]dihydrotetrabenazine (DTBZ) to quantify striatal monoaminergic innervation in patients with TS (n = 19) and control subjects (n = 27). Compartmental modeling was used to determine blood to brain ligand transport (K(1)) and tissue to plasma distribution volume (a measure of ligand binding) during continuous infusion of DTBZ. TS data were compared with control data using predefined regions of interest and on a voxel by voxel basis. There were no significant differences in ligand binding or ligand transport between patients with TS and control subjects in the dorsal striatum. With voxel by voxel analysis, there was increased DTBZ binding in the right ventral striatum. Previously reported differences between patients with TS and control subjects in dorsal striatal dopamine terminal markers may reflect medication-induced regulation of terminal marker expression or be the result of intrinsic differences in striatal dopaminergic synaptic function. Increased right ventral striatal DTBZ binding suggests that abnormal ventral striatal dopaminergic innervation may underlie tics.