Introduction

From Assay Guidance Wiki

Binding of GTP to the alpha subunit of heterotrimeric GTP binding proteins is an early event in agonist-induced activation of G-Protein-Coupled Receptors. Although GTPγS binding assays are carried out using membrane preparations in much the same way as radioligand binding assays, these are functional assays and can thus be used to differentiate agonist, antagonist, and inverse agonist activities. Such assays are carried out using [35S]guanosine-5’-O-(3thio)triphosphate which provides a radioactive ligand with high affinity for G-protein alpha subunits that is highly resistant to the inherent GTPase activity of alpha subunits such that it remains bound for sufficient periods of time to allow counting of radioactivity. Although the classical method used for GTPγS binding has been filtration of radiolabeled membranes, scintillation proximity assays (SPA) are much more convenient and allow the use of an antibody capture technique which permits determination of receptor-mediated activation of specific G-protein families. Thus there are two basic methods for running homogeneous SPA GTPγS binding assays in 96 well plate format: whole membrane binding in which labeled membranes are bound to wheat germ agglutinin (WGA)-coated SPA beads in the same way as these beads are used for radioligand binding assays, and antibody capture binding assays in which membranes are solubilized with detergent followed by isolation of the desired G-protein using a specific antibody along with capture of antibody-G-protein complexes onto anti-IgG coated SPA beads.

Advantages of GTPγS functional assays in comparison to determinations of second messengers produced as a result of receptor activation are:

1. The assays are very simple to run and utilize membrane preparations which can be frozen at -80°C for periods of months
2. Because GTP exchange is an event proximal to receptor activation these assays typically have lower degrees of receptor reserve than other functional assays and are thus useful for differentiating full from partial agonists
3. The assays are useful for determination of antagonist inhibition constants since agonists and antagonists can be equilibrated prior to starting the incubation by addition of GTPγ35S
4. One can often measure specific coupling of receptor subtypes to different G-protein families, even in native tissues, under very similar assay conditions

The major disadvantage is the relatively low signal to background which limits GTPγS binding to medium throughput evaluations. The power of the antibody capture technique is its ability to easily generate multiple concentration response curves thus allowing true pharmacological evaluation of receptor-mediated coupling to individual G-proteins, an accomplishment that is prohibitive by older immunoprecipitation techniques.