ABSTRACT
The human dopamine receptors D2S and D3 belong to the group of G protein-coupled receptors (GPCRs) and are important drug targets. Structural analyses and development of new receptor subtype specific drugs have been impeded by low expression yields or receptor instability. Fusing the T4 lysozyme into the intracellular loop 3 improves crystallization but complicates conformational studies. To circumvent these problems, we expressed the human D2S and D3 receptors in Escherichia coli using different N- and C-terminal fusion proteins and thermostabilizing mutations. We optimized expression times and used radioligand binding assays with whole cells and membrane homogenates to evaluate KD-values and the number of receptors in the cell membrane. We show that the presence but not the type of a C-terminal fusion protein is important. Bacteria expressing receptors capable of ligand binding can be selected using FACS analysis and a fluorescently labeled ligand. Improved receptor variants can thus be generated using error-prone PCR. Subsequent analysis of clones showed the distribution of mutations over the whole gene. Repeated cycles of PCR and FACS can be applied for selecting highly expressing receptor variants with high affinity ligand binding, which in the future can be used for analytical studies.
Subject(s)
Escherichia coli/genetics , Protein Engineering/methods , Receptors, Dopamine/genetics , Calibration , Cell Membrane/metabolism , Cloning, Molecular/methods , Escherichia coli/metabolism , Gene Library , Humans , Mutation , Organisms, Genetically Modified , Protein Engineering/standards , Receptors, Dopamine/metabolism , Receptors, Dopamine D2/genetics , Receptors, Dopamine D2/metabolism , Receptors, Dopamine D3/genetics , Receptors, Dopamine D3/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transformation, Bacterial , TransgenesABSTRACT
Cyclic adenosine monophosphate (cAMP) serves as a second messenger for numerous G-protein-coupled receptors. Changes in cellular cAMP levels reflect the biological activity of various GPCR-specific agents, including protein hormones. cAMP biosensors based on detection of Förster-type resonance energy transfer (FRET) offer unique advantages including the ratiometric nature of measurement, adjustable affinity toward detected molecule, capability of monitoring kinetics of cAMP release, and compatibility with the multi-well format and fluorescence plate reader platforms. In this chapter, we introduce the optimized version of the previously reported method to achieve sufficient and reproducible level of cAMP biosensor protein expression with the means of BacMam transduction system. As a practical challenge, we address the applicability of the designed assay for screening of biological activity of human hormones, including human chorionic gonadotropin (hCG) bearing different posttranslational modifications.
Subject(s)
Baculoviridae/metabolism , Chorionic Gonadotropin/metabolism , Cyclic AMP/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, LH/metabolism , Animals , Baculoviridae/genetics , Biosensing Techniques/methods , Cells, Cultured , Fluorescence Resonance Energy Transfer/methods , Humans , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Reproductive Control Agents/pharmacology , Signal TransductionABSTRACT
BRET and fluorescence anisotropy (FA) are two fluorescence-based techniques used for the characterization of ligand binding to G protein-coupled receptors (GPCRs) and both allow monitoring of ligand binding in real time. In this study, we present the first direct comparison of BRET-based and FA-based binding assays using the human M2 muscarinic acetylcholine receptor (M2R) and two TAMRA (5-carboxytetramethylrhodamine)-labeled fluorescent ligands as a model system. The determined fluorescent ligand affinities from both assays were in good agreement with results obtained from radioligand competition binding experiments. The assays yielded real-time kinetic binding data revealing differences in the mechanism of binding for the investigated fluorescent probes. Furthermore, the investigation of various unlabeled M2R ligands yielded pharmacological profiles in accordance with earlier reported data. Taken together, this study showed that BRET- and FA-based binding assays represent valuable alternatives to radioactivity-based methods for screening purposes and for a precise characterization of binding kinetics supporting the exploration of binding mechanisms.
Subject(s)
Fluorescent Dyes/chemistry , Receptor, Muscarinic M2/metabolism , Rhodamines/chemistry , Animals , Bioluminescence Resonance Energy Transfer Techniques , CHO Cells , Cricetulus , Fluorescence Polarization , HEK293 Cells , Humans , Ligands , Sf9 CellsABSTRACT
Dopamine receptors are G protein-coupled receptors that have several essential functions in the central nervous system. A better understanding of the regulatory mechanisms of ligand binding to the receptor may open new possibilities to affect the downstream signal transduction pathways. The majority of the available ligand binding assays use either membrane preparations, cell suspensions, or genetically modified receptors, which may give at least partially incorrect understanding of ligand binding. In this study, we implemented an assay combining fluorescence and bright-field microscopy to measure ligand binding to dopamine D3 receptors in live mammalian cells. For membrane fluorescence intensity quantification from microscopy images, we developed a machine learning-based user-friendly software membrane tools and incorporated it into a data management software aparecium that has been previously developed in our workgroup. For the experiments, a fluorescent ligand NAPS-Cy3B was synthesized by conjugating a dopaminergic antagonist N-(p-aminophenethyl)spiperone with a fluorophore Cy3B. The subnanomolar affinity of NAPS-Cy3B makes it a suitable ligand for the characterization of D3 receptors in live HEK293 cells. Using a microplate compatible automated widefield fluorescence microscope, together with the membrane tools software, enables the detection and quantification of ligand binding with a high-throughput. The live cell assay is suitable for the characterization of fluorescent ligand binding and also in the competition experiments for the screening of novel unlabeled dopaminergic ligands. We propose that this simple yet more native-like approach is feasible in GPCR research, as it enables the detection of ligand binding in an environment containing more components involved in the signal transduction cascade.
Subject(s)
Biological Assay , Carbocyanines/chemistry , Dopamine Antagonists/pharmacology , Receptors, Dopamine/metabolism , Software , Spiperone/analogs & derivatives , Dopamine/metabolism , Dopamine/pharmacology , Dopamine Antagonists/chemical synthesis , HEK293 Cells , Humans , Image Processing, Computer-Assisted/methods , Image Processing, Computer-Assisted/statistics & numerical data , Kinetics , Ligands , Machine Learning , Microscopy, Fluorescence/methods , Microscopy, Fluorescence/statistics & numerical data , Protein Binding , Spiperone/chemistryABSTRACT
Fluorescent ligands are versatile tools for the study of G protein-coupled receptors. Depending on the fluorophore, they can be used for a range of different applications, including fluorescence microscopy and bioluminescence or fluorescence resonance energy transfer (BRET or FRET) assays. Starting from phenylpiperazines and indanylamines, privileged scaffolds for dopamine D2-like receptors, we developed dansyl-labeled fluorescent ligands that are well accommodated in the binding pockets of D2 and D3 receptors. These receptors are the target proteins for the therapy for several neurologic and psychiatric disorders, including Parkinson's disease and schizophrenia. The dansyl-labeled ligands exhibit binding affinities up to 0.44 nM and 0.29 nM at D2R and D3R, respectively. When the dansyl label was exchanged for sterically more demanding xanthene or cyanine dyes, fluorescent ligands 10a-c retained excellent binding properties and, as expected from their indanylamine pharmacophore, acted as agonists at D2R. While the Cy3B-labeled ligand 10b was used to visualize D2R and D3R on the surface of living cells by total internal reflection microscopy, ligand 10a comprising a rhodamine label showed excellent properties in a NanoBRET binding assay at D3R.
Subject(s)
Carbocyanines/chemistry , Fluorescent Dyes/chemistry , Receptors, Dopamine D2 , Receptors, Dopamine D3 , Animals , CHO Cells , Cricetulus , HEK293 Cells , Humans , Receptors, Dopamine D2/chemistry , Receptors, Dopamine D2/genetics , Receptors, Dopamine D2/metabolism , Receptors, Dopamine D3/chemistry , Receptors, Dopamine D3/genetics , Receptors, Dopamine D3/metabolismABSTRACT
Dopamine receptors, which belong to the family of G protein-coupled receptors, are very substantial regulators in the brain and therefore important targets in drug discovery. Radioligand binding assay has been the method of choice for screening novel dopaminergic drugs for decades. We demonstrate that fluorescent ligand BodipyFL-SKF83566 binding to dopamine D1 receptors expressed in budded baculovirus particles can be characterized with fluorescent anisotropy (FA) based assay and that this is a valuable alternative to the radioligand binding assay. High binding affinity (KD = 5.2â¯nM) and fast association and dissociation kinetics (half-lives 40â¯s and 70â¯s, respectively) make BodipyFL-SKF83566 a suitable fluorescent ligand for this type of experiments. Good correlation between pKi values of 11 different dopaminergic ligands determined in competition binding experiments with radioligand [3H]SCH23390 or BodipyFL-SKF83566 (R2 = 0.96, slope not significantly different from unity) further validates the FA assay. In addition to competitor's affinity, the method also enables to quantify the apparent pIC50 values in time and hence kinetic properties of an unlabeled ligand can be estimated from the same competition binding experiment. Due to the homogenous nature of the FA assay reactions can be monitored in real time without any risk of precipitation of receptors in budded baculovirus particles. Despite the lack of coupled G proteins, the proposed novel assay system allows on-line monitoring of ligand binding to dopamine D1 receptors that could be easily applicable in early drug screening.
Subject(s)
Fluorescence Polarization , Receptors, Dopamine D1/metabolism , Animals , Baculoviridae/metabolism , Boron Compounds/metabolism , Ligands , Protein Binding , Sf9 Cells , SpodopteraABSTRACT
INTRODUCTION: G-protein-coupled receptors have become very important drug targets and therefore ligand binding assays for these receptors are an essential part of drug discovery. Among a variety of experimental systems, the radioligand binding assay has remained as one of the main methods in this field for decades. Usually cell membranes or tissues are used in these experiments, however in this article we demonstrate that baculoviruses produced in Sf9 cells display recombinant receptors on their surface and therefore can be used in radioligand binding assay. METHODS: We have used baculoviruses with dopamine D1 receptors as a model system to validate the usage of this receptor preparation in radioligand binding experiments. In order to collect membrane receptors and separate free radioligand, we have applied FilterMate Harvester with Filtermat B. RESULTS: Using baculoviruses with dopamine D1 receptors as a model system, we have shown that this is a suitable preparation for conventional radioligand binding assay. Here, all the experiments were performed using the dopamine D1-like receptor specific radioligand [3H]SCH23390. There were no significant differences between binding parameters determined in membranes of Sf9 cells and baculovirus particles. Similar pIC50 and Ki values were also determined in competition binding assays with HEK293 cell membranes. DISCUSSION: All the results obtained with baculovirus preparation were in good agreement with the data obtained in parallel experiments with membrane preparations from Sf9 and HEK293 cells expressing dopamine D1 receptors. Shape uniformity, homogeneous distribution and slow sedimentation of the membranes are some of the advantages of baculovirus preparations, which prove them as promising source of membrane proteins for routine and high throughput analysis.
Subject(s)
Baculoviridae/chemistry , Membrane Proteins/chemistry , Radioligand Assay/methods , Radiopharmaceuticals/chemistry , Receptors, Dopamine D1/drug effects , Virion/chemistry , Animals , Benzazepines/pharmacology , Binding, Competitive , Cell Line , Dopamine Agonists/pharmacology , Dopamine Antagonists/pharmacology , HEK293 Cells , Half-Life , Humans , Spodoptera/cytologyABSTRACT
During amniote evolution, the construction of the forebrain has diverged across different lineages, and accompanying the structural changes, functional diversification of the homologous brain regions has occurred. This can be assessed by studying the expression patterns of marker genes that are relevant in particular functional circuits. In all vertebrates, the dopaminergic system is responsible for the behavioral responses to environmental stimuli. Here we show that the brain regions that receive dopaminergic input through dopamine receptor D1 are relatively conserved, but with some important variations between three evolutionarily distant vertebrate lines-house mouse (Mus musculus), domestic chick (Gallus gallus domesticus) / common quail (Coturnix coturnix) and red-eared slider turtle (Trachemys scripta). Moreover, we find that in almost all instances, those brain regions expressing D1-like dopamine receptor genes also express Wfs1. Wfs1 has been studied primarily in the pancreas, where it regulates the endoplasmic reticulum (ER) stress response, cellular Ca2+ homeostasis, and insulin production and secretion. Using radioligand binding assays in wild type and Wfs1-/- mouse brains, we show that the number of binding sites of D1-like dopamine receptors is increased in the hippocampus of the mutant mice. We propose that the functional link between Wfs1 and D1-like dopamine receptors is evolutionarily conserved and plays an important role in adjusting behavioral reactions to environmental stimuli.
Subject(s)
Brain/metabolism , Gene Expression , Membrane Proteins/genetics , Receptors, Dopamine D1/metabolism , Animals , Biomarkers , Chick Embryo , Hippocampus/metabolism , Immunohistochemistry , Membrane Proteins/metabolism , Mice , Mice, Knockout , Protein Binding , Protein Transport , RNA, Messenger/genetics , Receptors, Dopamine D1/genetics , Receptors, Dopamine D5/genetics , Receptors, Dopamine D5/metabolismABSTRACT
Determination of biological activity of gonadotropin hormones is essential in reproductive medicine and pharmaceutical manufacturing of the hormonal preparations. The aim of the study was to adopt a G-protein coupled receptor (GPCR)-mediated signal transduction pathway based assay for quantification of biological activity of gonadotropins. We focussed on studying human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH), as these hormones are widely used in clinical practice. Receptor-specific changes in cellular cyclic adenosine monophosphate (cAMP, second messenger in GPCR signalling) were monitored by a Förster resonance energy transfer (FRET) biosensor protein TEpacVV in living cells upon activation of the relevant gonadotropin receptor. The BacMam gene delivery system was used for biosensor protein expression in target cells. In the developed assay only biologically active hormones initiated GPCR-mediated cellular signalling. High assay sensitivities were achieved for detection of hCG (limit of detection, LOD: 5 pM) and FSH (LOD: 100 pM). Even the small-scale conformational changes caused by thermal inactivation and reducing the biological activity of the hormones were registered. In conclusion, the proposed assay is suitable for quantification of biological activity of gonadotropins and is a good alternative to antibody- and animal-testing-based assays used in pharmaceutical industry and clinical research.
Subject(s)
Biosensing Techniques , Chorionic Gonadotropin/pharmacology , Fluorescence Resonance Energy Transfer , Follicle Stimulating Hormone/pharmacology , Cell Survival/drug effects , Cyclic AMP/metabolism , Humans , Recombinant Proteins/pharmacology , Time FactorsABSTRACT
Cyclic adenosine monophosphate (cAMP) is a second messenger of many G-protein-coupled receptors. The change in cellular cAMP level has widely been used to estimate the biological activity of various GPCR-specific agents. Förster resonance energy transfer (FRET) biosensors have been around for almost 10 years and became increasingly popular for cAMP detection. Ratiometric sensitized emission assay of a FRET biosensor can easily be implemented on fluorescence plate reader platforms. For such assays a considerable amount of cells expressing the desired biosensor is needed. A method to achieve sufficient and reproducible level of cAMP biosensor protein expression with the means of BacMam transduction system is the subject of this chapter.
Subject(s)
Baculoviridae/genetics , Biological Assay , Cyclic AMP/analysis , Gene Expression , Receptors, Dopamine D1/metabolism , Adenylyl Cyclases/metabolism , Animals , Baculoviridae/metabolism , Biosensing Techniques , Colforsin/pharmacology , Cyclic AMP/metabolism , Dopamine/pharmacology , Fluorescence Resonance Energy Transfer , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HEK293 Cells , Humans , Ligands , Plasmids/chemistry , Plasmids/metabolism , Receptors, Dopamine D1/agonists , Receptors, Dopamine D1/genetics , Second Messenger Systems , Sf9 Cells , Spodoptera , TransfectionABSTRACT
The present study for the first time is devoted to identify central effects of synthetic lunasin, a 43 amino acid peptide. A markedly expressed neuroleptic/cataleptic effect was observed at low (0.1-10 nmol/mouse) centrally administered doses in male C57Bl/6 mice. Lunasin considerably reduced the amphetamine hyperlocomotion but weakly apomorphine climbing behaviour. No influence on ketamine and bicuculline effects was observed. Binding assay studies demonstrated modest affinity of lunasin for the dopamine D1 receptor (Ki=60 ± 15 µM). In a functional assay of cAMP accumulation on live cells lunasin antagonised apomorphine effect on D1 receptor activation (pEC50=6.1 ± 0.3), but had no effect in cells expressing D2 receptors. The obtained data suggest that lunasin's action at least in part is provided via dopaminergic D1 receptor pathways. However, other non-identified mechanisms (probably intracellular) may play an important role in lunasin's central action. Nevertheless further studies of lunasin are promising, particularly taking into account a necessity for novel type of antipsychotic drugs.