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丁香实验推荐阅读
Immunoprecipitation and Phosphorylation of G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) are integral membrane proteins with seven transmembrane-spanning α-helices. Following ligand activation, many GPCRs are rapidly phosphorylated on serine/threonine residues in their cytoplasmic domains, principally the carboxyl ...

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Xenopus Oocyte Electrophysiology in GPCR Drug Discovery

Deorphanization of the large group of G protein-coupled receptors (GPCRs) for which an endogenous activating ligand has not yet been identified (orphan GPCRs) has become increasingly difficult. A specialized technique that has been successfully applied to deorphanize some of these ...

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A Quantum Dot-Labeled LigandReceptor Binding Assay for G Protein-Coupled Receptors Contained in Minimally Purified Membrane Nanopatches

A robust method to directly measure ligand–receptor binding interactions using fluorescence cross-correlation spectroscopy (FCCS) is described. The example receptor systems demonstrated here are the human mu-opioid receptor, a representative G protein-coupled recep ...

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Using Reporter Gene Technologies to Detect Changes in cAMP as a Result of GPCR Activation

The measurement of cyclic adenosine monophosphate levels through reporter gene technology represents one of the most popular and cost-effective methods to assess changes in functional activity of G protein-coupled receptors (GPCRs). This chapter provides a generic protocol for ...

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Detection of GPCR/-Arrestin Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer Technology

Bioluminescence resonance energy transfer (BRET) is a powerful and increasingly popular technique for studying protein–protein interactions in live cells and real time. In particular, there has been considerable interest in the ability to monitor interactions between G protei ...

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Application of Fluorescence Resonance Energy Transfer Techniques to Establish LigandReceptor Orientation

Fluorescence resonance energy transfer (FRET) has been utilized to determine distances between a fluorescence donor and a fluorescence acceptor having appropriately overlapping spectra. In this chapter, we utilize this approach to establish distances between a fluorescen ...

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Use of Fluorescence Indicators in Receptor Ligands

Fluorescence techniques can provide insights into the environment of fluorescent indicators incorporated within a ligand as it is bound to its receptor. Fluorescent indicators of different sizes and chemical characteristics ranging from hydrophilic to hydrophobic that are i ...

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New Insights into GPCR Function: Implications for HTS

G protein-coupled receptors (GPCRs) are a large family of proteins that represent targets for approximately 40% of all approved drugs. They possess unique structural motifs that allow them to interact with a diverse series of extracellular ligands, as well as intracellular signaling pr ...

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FLIPR Assays of Intracellular Calcium in GPCR Drug Discovery

Fluorescent dyes sensitive to changes in intracellular calcium have become increasingly popular in G protein-coupled receptor (GPCR) drug discovery for several reasons. First of all, the assays using the dyes are easy to perform and are of low cost compared to other assays. Second, most non-G ...

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FRET-Based Measurement of GPCR Conformational Changes

The C-termini of G protein-coupled receptors (GPCRs) interact with specific kinases and arrestins in an agonist-dependent manner suggesting that conformational changes induced by ligand binding within the transmembrane domains are transmitted to the C-terminus. F�rster res ...

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Resonant Waveguide Grating Biosensor for Whole-Cell GPCR Assays

Current drug discovery campaigns for G protein-coupled receptors (GPCRs) heavily rely on assay technologies that use artificial cell systems tailored to a point-of-contact readout and as a consequence are mostly pathway biased. Recently, we have developed label-free optical bios ...

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GPCR Microspot Assays on Solid Substrates

Multiplexing and miniaturization make microarrays an attractive tool for biomolecular interaction analysis. Adequate shelf life, mechanical stability through multiple assay steps, and amenability to a microplate format for screening are core requirements for the practi ...

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Yeast Assays for G Protein-Coupled Receptors

The functional coupling of heterologous G protein-coupled receptors (GPCRs) to the pheromone-response pathway of the budding yeast Saccharomyces cerevisiae is well established as an experimental system for ligand identification and for characterizing receptor pharmac ...

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BacMam: Versatile Gene Delivery Technology for GPCR Assays

BacMam viruses are modified baculoviruses that contain mammalian expression cassettes for viral gene delivery and transient expression in mammalian cells. They are easily, inexpensively, and rapidly generated and provide a versatile solution for G protein-coupled receptor ( ...

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Use of Aequorin for G protein-Coupled Receptor Hit Identification and Compound Profiling

G protein-coupled receptors (GPCRs) represent the largest class of targets in drug discovery, one-third of all marketed drugs are active at GPCRs and drugs targeted at GPCRs are marketed in virtually every therapeutic area. GPCRs can be classified by virtue of their coupling to second messen ...

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Use of the DiscoveRx Hithunter cAMPII Assay for Direct Measurement of cAMP in Gs and Gi GPCRs

G protein-coupled receptors (GPCRs) are druggable targets of great interest to the pharmaceutical industry. Generally, functional cell-based assays can be employed to detect agonists (inverse, partial, and full) in addition to allosteric and classical orthosteric antagonist ...

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A Time-Resolved Fluorescent Lanthanide (Eu)-GTP Binding Assay for Chemokine Receptors as Targets in Drug Discovery

Chemokines are a family of chemoattractant cytokines involved in leukocyte trafficking, activation, development, and hematopoeisis. Chemokines and their receptors have been implicated in several disease processes, particularly inflammatory and autoimmune disorde ...

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[35S]GTPS Binding in G Protein-Coupled Receptor Assays

The GTPγS binding assay to measure G protein activation following agonist binding to G protein-coupled receptors (GPCRs) remains a powerful molecular technique to substantiate traditional pharmacological values of potency, efficacy, and affinity. The method described uses m ...

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Teratology Studies in the Rat

The rat is the rodent species of choice for the regulatory safety testing of xenobiotics, such as medicinal products, food additives, and other chemicals. Many decades of experience and extensive data have accumulated for both general and developmental toxicology investigations in th ...

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The Teratology Testing of Cosmetics

In Europe, the developmental toxicity testing (including teratogenicity) of new cosmetic ingredients is performed according to the Cosmetics Directive 76/768/EEC: only alternatives leading to full replacement of animal experiments should be used. This chapter presents the th ...

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