Novel approaches to enhance the detection of receptor constitutive activity and inverse agonists

Abstract

Many wild-type G-protein-coupled receptors display relatively low levels of constitutive activity and thus identification of ligands with inverse agonist activity can be difficult. Two assays are available that monitor receptor interaction with a G protein, GTPase activity assays and [ 35S]GTPγS binding assays. GTPase assays are, however, frequently limited by high background. The fraction of membrane GTPase activity contributed by the constitutive activity of a receptor can be enhanced by addition of a recombinant regulator of G protein signalling protein to the assay. Detection of inverse agonists is then improved markedly. [ 35S]GTPγS binding assays for the detection of inverse agonists have historically been limited to receptors that activate pertussis toxin-sensitive G proteins. Introduction of a selective immunoprecipitation step using a G protein subtype specific antiserum allows this approach to be used for all G protein families. Application of these novel approaches, combined with the use of receptor–G protein fusion proteins to optimise receptor to G protein information transfer allows quantitative analysis of the extent of enhancement of constitutive activity produced by mutation of receptor or G protein.