Chapter Fifteen. Methods to Measure G-Protein-Coupled Receptor Activity for the Identification of Inverse Agonists
Department of Pharmacology, Faculty of Medicine, University of the Basque Country, Vizcaya, Spain. Methods in enzymology
(Impact Factor: 2.09).
12/2010; 485:261-73. DOI: 10.1016/B978-0-12-381296-4.00015-4
Before the concept of constitutive or intrinsic activity of the biological systems, which was formulated about thirty years ago, it was thought that agonist compounds were the only drugs capable of activating physiological responses, while antagonists were the ones capable of blocking them. However, this basic classification of drugs in pharmacology started to change only at the end of the eighties, when bioactive ligands, with negative efficacy, were developed. The G-protein-coupled receptors (GPCR) were promptly selected as one of the most useful types of pharmacological targets to study this inverse efficacy. This family of receptors is responsible for the signaling and control of many physiological processes, from the peripheral nervous system to the central. Therefore, the GPCR have become the most studied family of receptors in drug discovery. It has been estimated that around a third of the drugs actually used act via the GPCR, nevertheless there are still many orphan GPCR encoded by the human genome. During the last decade, reports and patents have described new methods to detect GPCR inverse agonist compounds. The detection of the G-protein constitutive activity and the quantification of the positive or negative efficacies induced by agonists or inverse agonists respectively has been studied by analyzing the binding of the nonhydrolyzable GTP analog, [(³⁵S]GTPγS. The present chapter describes an optimized method to detect GPCR inverse agonist ligands such as cannabinoid compounds, in both membrane homogenates and tissue sections (autoradiography).
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ABSTRACT: Because of their central role in the cellular response to growth factors, assays of MAP kinase activity are commonly used in pharmaceutical screening efforts aimed at detecting chemical modifiers of growth regulatory pathways. As our understanding of the complexity of signal transduction networks expands, however, it is becoming apparent that previously unappreciated temporal and contextual factors have profound effects on MAP kinase function. This is exemplified by recent studies of the regulation of the ERK1/2 MAP kinase cascade by GPCRs. Depending on receptor and cell type, GPCR stimulation of ERK1/2 can reflect a heterogenous array of signaling events. Activation of second messenger-dependent protein kinases and cross talk between GPCRs and receptor or nonreceptor tyrosine kinases can all induce ERK1/2 activation. Furthermore, a growing body of data indicates that the mechanism of ERK1/2 activation is a major determinant of ERK1/2 function. Activation of a nuclear pool of ERK1/2 as a consequence of cross talk between GPCRs and growth factor receptor tyrosine kinases may provide a mitogenic stimulus. In contrast, activation of ERK1/2 in localized pools on the membrane or confined to endosomal vesicles through the utilization of focal adhesions or beta-arrestins as "scaffolds" may spatially constrain ERK1/2 activity and favor the phosphorylation of nonnuclear ERK substrates. Findings such as these suggest that screening strategies that use single readouts of MAP kinase activity or function are likely to miss important signaling events, and point to the need for a multidimensional approach to MAP kinase-based screening efforts.
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ABSTRACT: Autoradiography is used to determine the anatomical distribution of biological molecules in human tissue and experimental animal models. This method is based on the analysis of the specific binding of radiolabeled compounds to locate neurotransmitter receptors or transporters in fresh frozen tissue slices. The anatomical resolution obtained by the quantification of the radioligands has allowed the density of receptor proteins to be mapped over the last forty years. The data yielded by autoradiography identify the receptors at their specific microscopic localization in the tissues and also in their native microenvironment, the intact cell membrane. Furthermore, in functional autoradiography the effects of small molecules on the activity of G protein-coupled receptors are evaluated. More recently, autoradiography has been combined with membrane microarrays to improve the high throughput screening of compounds. These technical advances have made autoradiography an essential analytical method for the progress of drug discovery. We include the future prospects and some preliminary results for imaging mass spectrometry (IMS) as a useful new method in pharmacodynamic and pharmacokinetic studies, complementing autoradiographic studies. IMS results could also be presented as density maps of molecules, proteins and metabolites in tissue sections, that can be identified, localized and quantified, with the advantage of avoiding any labeling of marker molecules. The limitations and future developments of these techniques are discussed here.
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ABSTRACT: The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention.
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