Use of the GTPγS ([35S]GTPγS and Eu-GTPγS) binding assay for analysis of ligand potency and efficacy at G protein-coupled receptors

Abstract

In this review I consider assays for G protein-coupled receptor (GPCR) activity based on the binding of labelled analogues of GTPγS ([(35) S]GTPγS or Eu-GTPγS) to G proteins in tissues (GTPγS binding assays). Such assays provide convenient measures of GPCR activity close to the receptor in the signalling cascade. In order to set up a GTPγS binding assay, the requirements of the assay must be considered. These are tissue source, GTPγS analogue, G protein, GDP, Mg(2+) /Na(+) ions, saponin, incubation time. The assay, once optimized, can be used to generate concentration/response curves for GPCRs signalling via G(i/o) proteins (or to other G proteins with a modified assay) and actions of agonists, inverse agonists and antagonists may, in principle, be assessed. For agonists and inverse agonists, data for the maximal agonist effect, the concentration of ligand giving a half-maximal response and the Hill coefficient may be derived. For antagonists, data for the equilibrium dissociation constant can be obtained. The mechanistic basis of the assay is considered. Although the assay can be used to profile ligands, under the conditions it is used, it may not be measuring the same event that determines GPCR action in cells.

Linked articles: This article is part of a themed section on Analytical Receptor Pharmacology in Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2010.161.issue-6

Figure 1

The G protein cycle. In panel A, the cycle is shown in the currently accepted form under cellular conditions ([GTP]∼50 µM). It should be noted that for G_i proteins the receptor/G protein (RG) complex may not dissociate into subunits (Bunemann et al., 2003; Frank et al., 2005). In panel B, the cycle is shown in the presence of a poorly hydrolysable analogue of GTP ([³⁵S]GTPγS), which increases the lifetime of the ARG.[³⁵S]GTPγS or Gα.[³⁵S]GTPγS species. The levels of GTPγS typically employed (∼0.1 nM in the [³⁵S]GTPγS binding assay) are very low compared with GTP levels under cellular conditions.