A Fully Automated [³⁵S]GTPγS Scintillation Proximity Assay for the High-Throughput Screening of G_i-Linked G Protein-Coupled Receptors

Abstract

The diversity of physiological functions mediated by the GPCR superfamily provides a rich source of molecular targets for drug discovery programs. Consequently, a variety of assays have been designed to identify lead molecules based on ligand binding or receptor function. In one of these, the binding of [(35)S]GTPgammaS, a nonhydrolyzable analogue of GTP, to receptor-activated G-protein alpha subunits represents a unique functional assay for GPCRs and is well suited for use with automated HTS. Here we compare [(35)S]GTPgammaS scintillation proximity binding assays for two different G(i)-coupled GPCRs, and describe their implementation with automated high-throughput systems.

Emerging Concepts of Guanine Nucleotide-Binding Protein-Coupled Receptor (GPCR) Function and Implications for High Throughput Screening

“…Simpler approaches used for HTS drug discovery involve measuring the ability of agonist to promote the binding of radiolabeled nonhydrolyzable GTP analogs, such as [ 35 S]GTP␥S, as a measure of GPCR activation. 150 Such binding can be detected using the scintillation proximity assay format and is easily adaptable for HTS. 151,152 Furthermore, nonradioactive alternatives exist to measure GTP␥S binding such as the DELFIA ® Eu-GTP␥S (PerkinElmer, Waltham, MA).…”

Biogenic amines and the control of neuromuscular signaling in schistosomes

“…As an alternative to cell-based signaling assays, researchers can measure receptor activation through the GTP/GDP exchange that occurs upon binding of an agonist. The procedure uses a radiolabeled non-hydrolyzable GTPcS analogue, which binds to Ga upon G-protein activation and is then quantified (Ferrer et al 2003;Eglen et al 2007). This assay was used by A. Maule and colleagues to identify a novel peptidergic GPCR from the planarian, Girardia tigrina, and to establish its agonist specificity (Omar et al 2007).…”

Receptor–ligand binding assays: Technologies and Applications

“…DMSO, MeOH up to 20% (v/v)) [29]. Immobilization of the receptor is based on either the interaction of glycoproteins and glycolipids with wheat germ agglutinin (WGA) [30][31][32][33], by capturing anti-receptor protein antibodies on an anti-IgG coated surface [34], via the streptavidin-biotin interaction [34] or via antibodies directed against the receptor [34,35].…”

“…The assay sensitivity in the Scintiplate is, due to the limited receptor binding capacity, lower as compared to the SPA beads [30], but can be improved by washing and drying the plate before measurement. These washing steps eliminate variations in counting efficiency as well as quenching, and moreover reduce non-specific binding [34].…”