Saturation Binding Analysis Research Articles

Novel Polystyrene-Binding Nanobody for Enhancing Immunoassays: Insights into Affinity, Immobilization, and Application Potential.

Nanobodies, which represent the next generation of antibodies due to their unique properties, face a significant limitation in their poor physical adsorption on solid supports. In this study, we successfully discovered polystyrene binding nanobodies from a synthetic nanobody library. Notably, bivalent nanobody B2 exhibited high affinity for polystyrene (0.7 nM for ELISA saturation binding analysis and 15.6 nM for isothermal titration calorimetry), displaying a pH-dependent behavior. Remarkably, hydrophobic and electrostatic interactions contribute minimally to the binding process. Molecular modeling provided insights into the interaction between B2 and polystyrene, revealing that the Trp51 residue within the CDR2 loop formed an aromatic H-bond with polystyrene at a distance of 2.74 Å, thus explaining the observed reduction in B2 affinity caused by Trp51 mutations. To explore B2's potential in protein immobilization, we constructed a bispecific nanobody by fusing B2 to an anticarcinoembryonic antigen nanobody 11C12, which cannot be immobilized on polystyrene through passive adsorption. Remarkably, the fusion construct achieved effective immobilization on polystyrene within 5 min by passing the need for periplasmic protein purification despite its low expression level. Moreover, the fusion construct demonstrated excellent linearity in the chemiluminescent enzyme immunoassay. For the first time, this study reports a simplified and seamless platform for the oriented immobilization of nanobody. Importantly, the entire process eliminated the need for protein purification, enabling efficient and rapid immobilization of fusion proteins directly from crude cell extracts, even when the expression level was low. Our developed process dramatically reduced the processing time from 2.5 days to just 5 min.

RO6807936 as a Novel PET Radiotracer for in Vitro and in Vivo Visualization and Quantification of BACE1 in the Rodent and Baboon Brain.

The beta-site amyloid precursor protein cleaving enzyme (BACE1) is responsible for initiating the generation of beta-amyloid, the major constituent of amyloid plaques in Alzheimer's disease (AD). The purpose of this study was to develop a specific BACE1 radioligand for visualization of the distribution pattern and quantification of the BACE1 protein in the rodent and monkey brain both in vitro by autoradiography and in vivo by positron emission tomography (PET). The BACE1 inhibitor RO6807936 originating from an in-house chemical drug optimization program was selected based on its PET tracer like physicochemical properties and a favorable pharmacokinetic profile. Saturation binding analysis of [3 H]RO6807936 revealed specific and high affinity binding (KD = 2.9 nM) and a low Bmax value (4.3 nM) of the BACE1 protein in native rat brain membranes. [3 H]RO6807936 binding showed a ubiquitous distribution on rat brain slices in vitro with higher levels in the CA3 8 cell layer and the granule cell layer of the hippocampus. In a next step, RO6807936 was successfully radiolabelled with carbon-11 and showed acceptable uptake in the baboon brain as well as a widespread and rather homogeneous distribution consistent with rodent data. In vivo blockade studies with a specific BACE1 inhibitor reduced uptake of the tracer to homogenous levels across brain regions and demonstrated specificity of the signal. Our data warrant further profiling of this PET tracer candidate in humans to investigate BACE1 expression in normal individuals and those with AD and as an imaging biomarker for target occupancy studies in clinical drug trials.

Evaluation of Serotonin 5‐HT 1A , 5‐HT 2A , and 5‐HT 2C Receptors and the Serotonin Transporter in an Fmr1 Knockout Mouse Model of Fragile X Syndrome

Fragile X syndrome (FXS)—caused by FMR1 gene silencing—is the leading monogenic cause of intellectual disability and autism spectrum disorder (ASD). Diminished serotonin (5-hydroxytryptamine, 5-HT) synthesis has been reported in ASD. The 5-HT transporter (SERT) and receptors (5-HTRs) have been targeted to manage ASD and FXS symptoms. Serotonergic neurons project to glutamate and GABA neurons expressing 5-HT1ARs, 5-HT2ARs, and 5-HT2CRs that modulate cellular excitation-inhibition which is altered in ASD and FXS. Here, we assessed the expression and function of these 5-HTRs and SERT in adult (P60-180) Fmr1 knockout (KO) mice, a model of FXS, compared to wild type (WT) mice. We performed autoradiography on brain sections using [3H]5-CT for 5-HT1ARs, [3H]Ketanserin for 5-HT2ARs, [3H]Mesulergine for 5-HT2CRs, [3H]Citalopram for SERT, and appropriate non-specific and off-target blocking ligands. Brain regions of interest were chosen and analyzed using ImageJ. Mean pixel intensity from the images was normalized to the mean of background intensity, and non-specific binding was subtracted from total binding to obtain normalized specific binding. Saturation binding assays, using whole brain membrane homogenates, were performed for 5-HT1ARs with [3H]5-CT and SB-269970 to block 5-HT7Rs; WAY-100635 was used to isolate 5-HT1AR specific binding. Behavioral effects of (R)-8-OH-DPAT, (±)-DOI, and lorcaserin were used to probe in vivo functional activity of 5-HT1A, 5-HT2A, and 5-HT2CRs, respectively. Responses to (R)-8-OH-DPAT (1 and 2 mg/kg), including flat body posture, head weaving, straub tail, hind limb abduction, piloerection, tremor, rearing, grooming, and head twitch responses (HTRs), were recorded by two independent observers. Locomotor effects of lorcaserin (1, 3, and 10 mg/kg) were recorded using Noldus Ethovision XT software, and HTRs elicited by (±)-DOI (1 mg/kg) were recorded using a hand-held tally counter. Sex effects were assessed in all tests. Preliminary results suggest that compared to WT males, Fmr1 KO males (N=3 per group) have higher 5-HT1AR expression in lateral septum and frontal cortex, lower 5-HT2AR expression in isocortex layer V, and lower 5-HT2CR expression in anterior olfactory nucleus and nucleus accumbens. Increased 5-HT1AR expression was confirmed by saturation binding analyses (N=6 per group). Male mice of both genotypes had lower 5-HT2CR expression in choroid plexus and caudate putamen (N=3 per group). Behavioral effects of (R)-8-OH-DPAT were significantly more pronounced in both male and female (N=5-7 per group) Fmr1 KO mice, relative to WT mice. A minor decrease in DOI-elicited HTRs was observed in Fmr1 KO males compared to WT males (14 vs. 17, N=8-9 per group). No genotype differences in locomotor activity were observed after lorcaserin administration (N=6 per group). Ongoing studies are assessing SERT expression and in vivo function via autoradiography and the tail suspension test, respectively. This study provides the first observations of perturbations in key 5-HTRs in Fmr1 KO mice, suggesting dysfunctions in the central 5-HT system in FXS.

The impact of 3,4-methylendioxymetamphetamine (MDMA) abstinence on seeking behavior and the expression of the D2-like and mGlu5 receptors in the rat brain using saturation binding analyses.

The abundance of research indicates that enriched environment acts as a beneficial factor reducing the risks of relapse in substance use disorder. There is also strong evidence showing the engagement of brain dopaminergic and glutamatergic signaling through the dopamine D2-like and metabotropic glutamate type 5 (mGlu5) receptors, respectively, that has a direct impact on drug reward and drug abstinence. The present study involved 3,4-methylendioxymethamphetamine (MDMA) self-administration with the yoked-triad procedure in rats kept under different housing conditions during abstinence - enriched environment (EE) or isolation cage (IC) conditions - aimed at evaluating changes in brain receptors affecting drug-seeking behavior as well as density and affinity of the D2-like and mGlu5 receptors in several regions of the animal brain. Our results show that exposure to EE conditions strongly reduced active lever presses during cue-induced drug-seeking. At the neurochemical level, we demonstrated marked decreases of D2-like receptor affinity in the dorsal striatum in rats previously self-administering MDMA under EE and increases in density under IC conditions. Moreover, we found the increases in the density and decreases in the affinity of the D2-like receptor in the prefrontal cortex and nucleus accumbens provoked by IC conditions. The mGlu5 receptor density decreased only in the prefrontal cortex after IC and EE abstinence. Moreover, our study has revealed a clear decrease in mGlu5 receptor density in the nucleus accumbens in the group actively administering MDMA only under EE conditions. This study demonstrates that housing conditions have impact on drug-seeking behavior in rats during abstinence from MDMA self-administration. The observed changes in the dopamine D2-like and mGlu5 receptor Bmax and/or Kd values were brain-region specific and related to either pharmacological and/or motivational features of MDMA.

Identification of a high affinity binding site for abscisic acid on human lanthionine synthetase component C-like protein 2.

Lanthionine synthetase component C-like protein 2 (LANCL2) has been identified as the mammalian receptor mediating the functional effects of the universal stress hormone abscisic acid (ABA) in mammals. ABA stimulates insulin independent glucose uptake in myocytes and adipocytes via LANCL2 binding in vitro, improves glucose tolerance in vivo and induces brown fat activity in vitro and in vivo. The emerging role of the ABA/LANCL2 system in glucose and lipid metabolism makes it an attractive target for pharmacological interventions in diabetes mellitus and the metabolic syndrome. The aim of this study was to investigate the presence of ABA binding site(s) on LANCL2 and identify the amino acid residues involved in ABA binding. Equilibrium binding assays ([3H]-ABA saturation binding and surface plasmon resonance analysis) suggested multiple ABA-binding sites, prompting us to perform a computational study that indicated one putative high-affinity and two low-affinity binding sites. Site-directed mutagenesis (single mutant R118I, triple mutants R118I/R22I/K362I and R118I/S41A/E46I) and equilibrium binding experiments on the mutated LANCL2 proteins identified a high-affinity ABA-binding site involving R118, with a KD of 2.6 nM ± 1.2 nM, as determined by surface plasmon resonance. Scatchard plot analysis of binding curves from both types of equilibrium binding assays revealed a Hill coefficient >1, suggesting cooperativity of ABA binding to LANCL2. Identification of the high-affinity ABA-binding site is expected to allow the design of ABA agonists/antagonists, which will help to understand the role of the ABA/LANCL2 system in human physiology and disease.

Immobilization methods for the rapid total chemical synthesis of proteins on microtiter plates.

The chemical synthesis of proteins typically involves the solid-phase peptide synthesis of unprotected peptide fragments that are stitched together in solution by native chemical ligation (NCL). The process is slow, and throughput is limited because of the need for repeated high performance liquid chromatography purification steps after both solid-phase peptide synthesis and NCL. With an aim to provide faster access to functional proteins and to accelerate the functional analysis of synthetic proteins by parallelization, we developed a method for the high performance liquid chromatography-free synthesis of proteins on the surface of microtiter plates. The method relies on solid-phase synthesis of unprotected peptide fragments, immobilization of the C-terminal fragment and on-surface NCL with an unprotected peptide thioester in crude form. Herein, we describe the development of a suitable immobilization chemistry. We compared (i) formation of nickel(II)-oligohistidine complexes, (ii) Cu-based [2+3] alkine-azide cycloaddition and (iii) hydrazone ligation. The comparative study identified the hydrazone ligation as most suitable. The sequence of immobilization via hydrazone ligation, on-surface NCL and radical desulfurization furnished the targeted SH3 domains in near quantitative yield. The synthetic proteins were functional as demonstrated by an on-surface fluorescence-based saturation binding analysis. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Histamine type 1-receptor activation by low dose of histamine undermines human glomerular slit diaphragm integrity

Histamine has been reported to decrease the ultrafiltration coefficient, which inversely correlates with glomerular permselectivity, however the mechanism(s) underling this effect have never been investigated. This study aimed to assess whether histamine could exert a direct detrimental effect on podocyte permeability and the possible involvement of two key proteins for the glomerular slit diaphragm (SD) integrity, zonula occludens-1 (ZO-1) and P-cadherin.The effect of histamine (100 pM–1000nM) on coloured podocytes junctional integrity was evaluated functionally by a transwell assay of monolayer permeability and morphologically by electron microscopy. Histamine receptor (H1-4R) presence was evaluated at both mRNA (RT-PCR) and protein (immunofluorescence) levels. The Kd and Bmax values for [3H]mepyramine were determined by saturation binding analysis; IP1 and cAMP production evoked by histamine were measured by TR-FRET. ZO-1, P-cadherin and vimentin expression was assessed by qRT-PCR and quantitative immunoblotting.Histamine elicited a time- and sigmoidal dose-dependent (maximum effect at 8h, 10nM) increase in podocyte paracellular permeability widening the paracellular spaces. Only H1R was predominantly localised to the podocyte membrane. Consistently, histamine elicited a sigmoidal dose-dependent increase in IP1, but not in cAMP. Histamine exposure evoked a concentration-dependent reduction in both ZO-1 and P-cadherin and a parallel induction of vimentin mRNA expression with a maximum effect after 6h, and protein expression with a maximum effect after 8h. These effects were prevented by the selective H1R antagonist chlorpheniramine.In conclusion, our data demonstrate that histamine, via the H1R, modifies SD morphological and functional integrity, in part, by decreasing the expression of ZO-1 and P-cadherin.

Distribution and Estrogen Regulation of Membrane Progesterone Receptor-β in the Female Rat Brain

Although several studies have reported the localization of membrane progesterone (P(4)) receptors (mPR) in various tissues, few have attempted to describe the distribution and regulation of these receptors in the brain. In the present study, we investigated expression of two mPR subtypes, mPRα and mPRβ, within regions of the brain, known to express estradiol (E(2))-dependent [preoptic area (POA) and hypothalamus] and independent (cortex) classical progestin receptors. Saturation binding and Scatchard analyses on plasma membranes prepared from rat cortex, hypothalamus, and POA demonstrated high-affinity, specific P(4)-binding sites characteristic of mPR. Using quantitative RT-PCR, we found that mPRβ mRNA was expressed at higher levels than mPRα, indicating that mPRβ may be the primary mPR subtype in the rat brain. We also mapped the distribution of mPRβ protein using immunohistochemistry. The mPRβ-immunoreactive neurons were highly expressed in select nuclei of the hypothalamus (paraventricular nucleus, ventromedial hypothalamus, and arcuate nucleus), forebrain (medial septum and horizontal diagonal band), and midbrain (oculomotor and red nuclei) and throughout many areas of the cortex and thalamus. Treatment of ovariectomized female rats with E(2) benzoate increased mPRβ immunoreactivity within the medial septum but not the medial POA, horizontal diagonal band, or oculomotor nucleus. Together, these findings demonstrate a wide distribution of mPRβ in the rodent brain that may contribute to functions affecting behavioral, endocrine, motor, and sensory systems. Furthermore, E(2) regulation of mPRβ indicates a mechanism through which estrogens can regulate P(4) function within discrete brain regions to potentially impact behavior.

Changes in hippocampal GABA A/cBZR density during limbic epileptogenesis: Relationship to cell loss and mossy fibre sprouting

Reduced GABA A/central benzodiazepine receptor (GABA A/cBZR) density, mossy fibre sprouting (MFS) and hippocampal cell loss are well described pathological features of human temporal lobe epilepsy (TLE), and animal models thereof. However, the temporal relationship of their development, and their roles in the emergence of the epilepsy, are uncertain. This was investigated in the kainic acid (KA)-induced post-status epilepticus (SE) model of TLE. Male Wistar rats (7 weeks, n = 53) were randomised into control and KA groups. At 24 h, 2, 4 or 6 weeks sham and KA post-SE animals were euthanised, brains extracted and GABA A/cBZR density, neuronal loss and MFS measured in hippocampal sub-regions. GABA A/cBZR density ( B max) was measured by saturation-binding analysis using [ 3H]-flumazenil. At 24 h post-SE GABA A/cBZR density was increased in almost all hippocampal subregions, but was decreased at the later time points with the exception of the dentate gyrus. There was significant neuronal loss in the CA3 SPc region (−24±9.3%, p<0.05) at 24h, which remained stable at the later time points associated with an elevated GABA A/cBZR density per surviving neuron at 24h post-SE (+56.4%; p<0.05) which returned to control levels by 6 weeks post-SE. MFS in the dentate gyrus progressively increased over the 6 weeks following SE (+ 70.6% at 6 weeks), at which time there was a significant inverse relationship with GABA A/cBZR binding ( r 2 = 0.87; p = 0.02). The temporal evolution of GABA A/cBZR density changes post-KA-induced SE, and the relationship with decreases in hippocampal pyramidal cell numbers and MFS, may point to a key role for these changes in the pathogenesis of acquired limbic epileptogenesis.

Quantification of D2 Receptor Binding in the Rat Striatum Using Small Animal PET - The Impact of the Reference Tissue on the Binding Potential of [18F]N-Methyl-Benperidol

In the present study, the binding potential (BP) of the D2 receptor radioligand ( 18 F)N-methyl-benperidol (( 18 F)FMB) was determined with in vivo saturation binding analysis using either cerebellum or parietal cortex as reference region. For the purpose of validation, BP additionally was determined in the same set of animals by computing the equilibrium ratios of the distribution volumes (V3''). With in vivo saturation binding analysis, the striatal BP as obtained with a cortical estimate for the free and non-specifically bound radioligand fell short of the BP as obtained with a cerebellar estimate by 70%. Similarly, with the equilibrium distribution volume method, the employment of a cortical REF led to a striatal V3'', which was 30% lower than the V3'' obtained with a cerebellar REF. The BP determined with in vivo saturation binding analysis and a cerebellar REF provided a closer approximation to the BP previously obtained with in vitro autoradiography relative to the BP determined with in vivo saturation binding analysis and a cortical REF. Findings show that in vivo saturation binding analysis is a feasible approach to obtain receptor binding parameters if scanner characteristics or other technical limitations preclude the kinetic modelling of binding data. However, caution should be exerted in choosing the reference tissue with regard to the intended investigation.

Declines of hippocampal metabotropic glutamate receptor in Alzheimer's disease

Glutamatergic networks subserve the vast majority of motor, sensory and integrative brain functions. In these networks, metabotropic glutamate receptor type five (mGLUR5) modulates plastic changes associated with efficiency of glutamate neurotransmission. Pre-clinical studies strongly suggest that mGLUR5 plays a role in Alzheimer's disease. Human hippocampi tissue were obtained from from AD patients (N = 5, age 72 ± 4.39) and healthy controls (N = 5, age 69.67 ± 5.61) (Brain Bank of Douglas Hospital, Montreal, Canada). The tissue was cryosectioned (20 μm thick) and a saturation binding study was carried out. The slides were pre-incubated for 20 min in buffer containing 30 mM Na HEPES, 110 nM NaCl, 5 mM KCl, 2.5 mM CaCl2 and 1.2 mM MgCl2 (pH 7.4) and then incubated for 60 min in the presence of [3H]ABP688 (74 Ci/mmol) at a range of concentrations from 0.125 to 8 nM. Non-specific binding was determined with addition of 10 μM of the mGLUR5 antagonist MPEP in adjacent sections. After the incubation, the slides were washed (3 x 5 min) in cold buffer, dipped in ice-cold distilled water and dried. The sections along with autoradiographic standards were exposed for five days to phosphor imaging plates. The Bmax and KD of [3H]ABP688 from both groups were calculated by saturation binding analysis and compared using F-test (p < 0.05). Saturation binding using [3H]ABP688 revealed higher Bmax in the control group (Ctrl, 1.31 ± 0.06; AD 0.711 ± 0.03 pmol/mg of tissue). Binding of [3H]ABP688 in human hippocampal cryosections was blocked after addition of 10 μM MPEP (Figure 1). The nearly 50% reduction in total concentration of receptors in the AD hippocampi was statistically significant (p < 0.001). The KD (1.43 ± 0.2 nM) did not change across groups. Our results indicate mGLUR5 vulnerability in AD. These results support the use of mGLUR5 PET molecular agents for imaging glutamatergic abnormalities in AD patients.

Progesterone Receptor A (PRA) and PRB-Independent Effects of Progesterone on Gonadotropin-Releasing Hormone Release

Progesterone's (P4) negative feedback actions in the female reproductive axis are exerted in part by suppression of hypothalamic GnRH release. Here we show that P4 can inhibit GnRH release by a mechanism independent of a nuclear P4 receptor (PR(A/B)). Injections of P4, but not vehicle, allopregnanolone, or dexamethasone, acutely suppressed LH levels in both wild-type and P4 receptor knockout ovariectomized mice; pituitary responsiveness to GnRH was retained during P4 treatment, indicating a hypothalamic action. Superfusion of GnRH-producing GT1-7 cells with medium containing 10(-7) m P4 produced a rapid reduction in GnRH release. Incubation with P4 (10(-9) to 10(-7) M) inhibited forskolin-stimulated cAMP accumulation; cotreatment with pertussis toxin prevented this effect. Treatment of GT1-7 cell membranes with P4 caused activation of an inhibitory G protein (G(i)), as shown by immunoprecipitation with a G(i) antibody of most of the increase in membrane-bound [(35)S]GTPgamma-S. Saturation binding analyses demonstrated the presence of a high affinity (K(d) 5.85 nM), limited capacity (Bmax 62.2 nM) binding site for P4. RT-PCR analysis revealed the presence of mRNAs encoding both isoforms of the membrane P4 receptors, mPRalpha and mPRbeta. Western blotting, immunocytochemistry, and flow cytometry experiments similarly revealed expression of mPR proteins in the plasma membranes of GT1-7 cells. Treatment with mPRalpha siRNA attenuated specific P4 binding to GT1-7 cell membranes and reversed the P4 inhibition of cAMP accumulation. Taken together, our results suggest that negative feedback actions of P4 include rapid PR(A/B)-independent effects on GnRH release that may in part be mediated by mPRs.

Epitope-tagged Receptor Knock-in Mice Reveal That Differential Desensitization of α2-Adrenergic Responses Is because of Ligand-selective Internalization

Although ligand-selective regulation of G protein-coupled receptor-mediated signaling and trafficking are well documented, little is known about whether ligand-selective effects occur on endogenous receptors or whether such effects modify the signaling response in physiologically relevant cells. Using a gene targeting approach, we generated a knock-in mouse line, in which N-terminal hemagglutinin epitope-tagged alpha(2A)-adrenergic receptor (AR) expression was driven by the endogenous mouse alpha(2A)AR gene locus. Exploiting this mouse line, we evaluated alpha(2A)AR trafficking and alpha(2A)AR-mediated inhibition of Ca(2+) currents in native sympathetic neurons in response to clonidine and guanfacine, two drugs used for treatment of hypertension, attention deficit and hyperactivity disorder, and enhancement of analgesia through actions on the alpha(2A)AR subtype. We discovered a more rapid desensitization of Ca(2+) current suppression by clonidine than guanfacine, which paralleled a more marked receptor phosphorylation and endocytosis of alpha(2A)AR evoked by clonidine than by guanfacine. Clonidine-induced alpha(2A)AR desensitization, but not receptor phosphorylation, was attenuated by blockade of endocytosis with concanavalin A, indicating a critical role for internalization of alpha(2A)AR in desensitization to this ligand. Our data on endogenous receptor-mediated signaling and trafficking in native cells reveal not only differential regulation of G protein-coupled receptor endocytosis by different ligands, but also a differential contribution of receptor endocytosis to signaling desensitization. Taken together, our data suggest that these HA-alpha(2A)AR knock-in mice will serve as an important model in developing ligands to favor endocytosis or nonendocytosis of receptors, depending on the target cell and pathophysiology being addressed.

The C-terminal Tail of CRTH2 Is a Key Molecular Determinant That Constrains Gαi and Downstream Signaling Cascade Activation

Prostaglandin D(2) activation of the seven-transmembrane receptor CRTH2 regulates numerous cell functions that are important in inflammatory diseases, such as asthma. Despite its disease implication, no studies to date aimed at identifying receptor domains governing signaling and surface expression of human CRTH2. We tested the hypothesis that CRTH2 may take advantage of its C-tail to silence its own signaling and that this mechanism may explain the poor functional responses observed with CRTH2 in heterologous expression systems. Although the C terminus is a critical determinant for retention of CRTH2 at the plasma membrane, the presence of this domain confers a signaling-compromised conformation onto the receptor. Indeed, a mutant receptor lacking the major portion of its C-terminal tail displays paradoxically enhanced Galpha(i) and ERK1/2 activation despite enhanced constitutive and agonist-mediated internalization. Enhanced activation of Galpha(i) proteins and downstream signaling cascades is probably due to the inability of the tail-truncated receptor to recruit beta-arrestin2 and undergo homologous desensitization. Unexpectedly, CRTH2 is not phosphorylated upon agonist-stimulation, a primary mechanism by which GPCR activity is regulated. Dynamic mass redistribution assays, which allow label-free monitoring of all major G protein pathways in real time, confirm that the C terminus inhibits Galpha(i) signaling of CRTH2 but does not encode G protein specificity determinants. We propose that intrinsic CRTH2 inhibition by its C terminus may represent a rather unappreciated strategy employed by a GPCR to specify the extent of G protein activation and that this mechanism may compensate for the absence of the classical phosphorylation-dependent signal attenuation.

Development of a competitive binding assay system with recombinant estrogen receptors from multiple species

In the current study, we developed a new system using full-length recombinant baculovirus-expressed estrogen receptors which allows for direct comparison of binding across species. Estrogen receptors representing five vertebrate classes were compared: human estrogen receptor alpha (hERα), quail estrogen receptor alpha (qERα), alligator estrogen receptor alpha (aERα), salamander estrogen receptor alpha (sERα), and fathead minnow estrogen receptor alpha (fhERα). Saturation binding analyses indicated 17β-estradiol (E2) dissociation constants (Kd) were 0.22 ± 0.02 nM for hERα, 0.28 ± 0.04 nM for sERα, 0.44 ± 0.04 nM for aERα, 0.58 ± 0.10 nM for qERα, and 0.58 ± 0.05 nM for fhERα. Binding specificity to each of the receptors was evaluated using E2, dihydrotestosterone (DHT), corticosterone (C), and ethinylestradiol (EE). E2 and EE were strong binders in all species with IC50's ranging from 0.65 nM with hERα to 1.01 nM with sERα for E2 and from 0.68 nM with sERα to 1.20 nM with qERα for EE. DHT was a weak binder with IC50's ranging from 3.3 μM with hERα to 39 μM with fhERα, and C did not bind any of the receptors at concentrations up to 100 μM. This system provides a convenient in vitro approach for directly comparing chemical binding to estrogen receptors across multiple species without the need to sacrifice animals.

P.1.c.020 Chronic treatment with imidazoline I2-drugs down-regulates key proteins of the canonical apoptotic pathways in rat brain

[3H]2-(2-benzofuranyl)-2-imidazoline (2-BFI) and [3H]idazoxan are the most used tools to characterise imidazoline I2 receptors. We evaluated the binding of both radioligands to human postmortem frontal cortex membranes. Saturation binding analyses revealed that [3H]idazoxan (in the presence of 2 μM efaroxan to avoid radioligand binding to α2-adrenoceptors and imidazoline I1 receptors) and [3H]2-BFI bound with high affinity to an apparent single population of sites. However, in competition studies whereas [3H]idazoxan (10 nM) binding was displaced monophasically by idazoxan and 2-BFI, both drugs displayed biphasic curves for [3H]2-BFI (1 nM). The proportion of the low-affinity binding site increased from 17% to 25% when 10 nM [3H]2-BFI was displaced by idazoxan. Amiloride inhibited [3H]2-BFI (10 nM) binding with low affinity and in a monophasic way. These data indicate that [3H]2-BFI recognises in human postmortem brain membranes a second binding site different from the imidazoline I2 receptors labelled by [3H]idazoxan.

Effect of sphingomyelinase treatment on ligand binding activity of human serotonin 1A receptors

The serotonin 1A receptor is an important member of the G-protein coupled receptor family, and is involved in the generation and modulation of a variety of cognitive, behavioral, and developmental functions. We have monitored the ligand binding of the human serotonin 1A receptor stably expressed in CHO cells (termed CHO-5-HT 1AR) following treatment with sphingomyelinase (SMase), an enzyme that specifically catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine. Our results show, for the first time, that the specific ligand binding activity of the serotonin 1A receptor in membranes isolated from CHO-5-HT 1AR cells is increased upon sphingomyelinase treatment. Saturation binding analysis reveals increase in binding affinity of the receptor under these conditions. This is accompanied by a reduction in membrane order, as monitored by fluorescence anisotropy of the membrane probe 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) in intact cells. These results represent the first report on the effect of sphingomyelinase treatment on the ligand binding activity of this important neurotransmitter receptor.

Constitutive Activity of the Cannabinoid CB1 Receptor Regulates the Function of Co-expressed Mu Opioid Receptors

The human mu opioid receptor was expressed stably in Flp-In T-REx HEK293 cells. Occupancy by the agonist DAMGO (Tyr-d-Ala-Gly-N-methyl-Phe-Gly-ol) resulted in phosphorylation of the ERK1/2 MAP kinases, which was blocked by the opioid antagonist naloxone but not the cannabinoid CB1 receptor inverse agonist SR141716A. Expression of the human cannabinoid CB1 receptor in these cells from the inducible Flp-In T-REx locus did not alter expression levels of the mu opioid receptor. This allowed the cannabinoid CB1 agonist WIN55212-2 to stimulate ERK1/2 phosphorylation but resulted in a large reduction in the capacity of DAMGO to activate these kinases. Although lacking affinity for the mu opioid receptor, co-addition of SR141716A caused recovery of the effectiveness of DAMGO. In contrast co-addition of the CB1 receptor neutral antagonist O-2050 did not. Induction of the CB1 receptor also resulted in an increase of basal [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and thereby a greatly reduced capacity of DAMGO to further stimulate [(35)S]GTPgammaS binding. CB1 inverse agonists attenuated basal [(35)S]GTPgammaS binding and restored the capacity of DAMGO to stimulate. Flp-In T-REx HEK293 cells were generated, which express the human mu opioid receptor constitutively and harbor a modified D163N cannabinoid CB1 receptor that lacks constitutive activity. Induction of expression of the modified cannabinoid CB1 receptor did not limit DAMGO-mediated ERK1/2 MAP kinase phosphorylation and did not allow SR141716A to enhance the function of DAMGO. These data indicate that it is the constitutive activity inherent in the cannabinoid CB1 receptor that reduces the capacity of co-expressed mu opioid receptor to function.

Amyloid toxicity in skeletal myoblasts: Implications for inclusion-body myositis

Skeletal muscle disorder, inclusion-body myositis (IBM) has been known for accumulation of amyloid characteristic proteins in muscle. To understand the biophysical basis of IBM, the interaction of amyloid fibrils with skeletal myoblast cells (SMC) has been studied in vitro. Synthetic insulin fibrils and Aβ25–35 fibrils were used for this investigation. From the saturation binding analysis, the calculated dissociation constant (Kd) for insulin fibril and Aβ25–35 fibrils were 69.37±11.17nM and 115.60±12.17nM, respectively. The fibrillar insulin comparatively has higher affinity binding to SMC than Aβ fibrils. The competitive binding studies with native insulin showed that the amount of bound insulin fibril was significantly decreased due to displacement of native insulin. However, the presence of native insulin is not altered the binding of β-amyloid fibril. The cytotoxicity of insulin amyloid intermediates was measured. The pre-fibrillar intermediates of insulin showed significant toxicity (35%) as compared to matured fibrils. Myoblast treated with β-amyloid fibrils showed more oxidative damage than the insulin fibril. Cell differentiating action of amyloidic insulin was assayed by creatine kinase activity. The insulin fibril treated cells differentiated more slowly compared to native insulin. However, β-amyloid fibrils do not show cell differentiation property. These findings reinforce the hypothesis that accumulation of amyloid related proteins is significant for the pathological events that could lead to muscle degeneration and weakness in IBM.

Competitive binding comparison of endocrine‐disrupting compounds to recombinant androgen receptor from fathead minnow, rainbow trout, and human

Typically, in vitro hazard assessments for the identification of endocrine-disrupting compounds (EDCs), including those outlined in the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Tier 1 Screening protocols, utilize mammalian receptors. Evidence, however, exists that fish sex steroid hormone receptors differ from mammalian receptors both structurally and in their binding affinities for some steroids and environmental chemicals. Most of the binding studies to date have been conducted using cytosolic preparations from various tissues. In the present study, we compare competitive binding of a set of compounds to full-length recombinant rainbow trout androgen receptor alpha (rtAR), fathead minnow androgen receptor (fhAR), and human androgen receptor (hAR), each expressed in COS cells. Saturation binding and subsequent Scatchard analysis using [3H]R1881, a high-affinity synthetic androgen, revealed an equilibrium dissociation constant (Kd) of 0.11 nM for the rtAR, 1.8 nM for the fhAR, and 0.84 nM for the hAR. Compounds, including endogenous and synthetic steroids, known mammalian antiandrogens, and environmental compounds, were tested for competitive binding to each of the three receptors. Overall, agreement existed across receptors as to binding versus nonbinding for all compounds tested in this study. Minor differences, however, were found in the relative order of binding of the compounds to the individual receptors. Studies such as these will facilitate the identification of EDCs that may differentially affect specific species and aid in the development and support of future risk assessment protocols.