Elsevier

Life Sciences

Volume 74, Issue 4, 12 December 2003, Pages 489-508
Life Sciences

The [35S]GTPγS binding assay: approaches and applications in pharmacology

https://doi.org/10.1016/j.lfs.2003.07.005Get rights and content

Abstract

Receptors of the of seven transmembrane spanning, heterotrimeric G protein coupled family (GPCR) play crucial roles in regulating physiological functions and consequently are targets for the action of many classes of drugs. Activation of receptor by agonist leads to the dissociation of GDP from Gα of the Gαβγ heterotrimer, followed by the binding of GTP to Gα and subsequent modulation of downstream effectors. The G protein heterotrimer is reformed by GTPase activity of the Gα subunit, forming Gα-GDP and so allowing Gα and Gβγ to recombine. The [35S]GTPγS assay measures the level of G protein activation following agonist occupation of a GPCR, by determining the binding of the non-hydrolyzable analog [35S]GTPγS to Gα subunits. Thus, the assay measures a functional consequence of receptor occupancy at one of the earliest receptor-mediated events. The assay allows for traditional pharmacological parameters of potency, efficacy and antagonist affinity, with the advantage that agonist measures are not subjected to amplification or other modulation that may occur when analyzing parameters further downstream of the receptor. In general the assay is experimentally more feasible for receptors coupled to the abundant Gi/o proteins. Nevertheless, [35S]GTPγS binding assays are used with GPCRs that couple to the Gs and Gq families of G proteins, especially in artificial expression systems, or using receptor-Gα constructs or immunoprecipitation of [35S]GTPγS-labeled Gα. The relative simplicity of the assay has made it very popular and its use is providing insights into contemporary pharmacological topics including the roles of accessory proteins in signaling, constitutive activity of receptors and agonist specific signaling.

Introduction

The [35S]GTPγS binding assay measures the level of G protein activation following agonist occupation of a G protein-coupled receptor (GPCR). The great advantage of the assay is that it measures a functional consequence of receptor occupancy at one of the earliest receptor-mediated events. This means that it can be used to provide traditional pharmacological parameters of potency, efficacy and antagonist affinity, with the advantage that agonist measures are not subjected to amplification or other modulation that may occur when analyzing parameters further downstream of the receptor. More recent techniques allow this assay to identify the particular G protein(s) to which a receptor couples, to study agonist-directed trafficking and to be used in a high throughput format.

Section snippets

G protein coupled receptors and G proteins

Members of the family of seven transmembrane spanning, heterotrimeric G protein coupled receptors bind diverse ligands including neurotransmitters, hormones, peptides and nucleotides. As such, GPCRs play crucial roles in regulating physiological functions and consequently are targets for many drugs including analgesics, antihistamines, neuroleptics, antidepressants, anti-asthmatics and drugs for cardiovascular disorders. Structurally, GPCRs consist of an extracellular N terminus, seven

The [35S]GTPγS binding assay

The [35S]GTPγS binding assay measures the level of G protein activation following agonist occupation of a GPCR. In the assay [35S]GTPγS replaces endogenous GTP and binds to the Gα subunit following activation of the receptor to form a Gα-[35S]GTPγS species. Since the γ-thiophosphate bond is resistant to hydrolysis by the GTPase of Gα, G protein is prevented from reforming as a heterotrimer and thus [35S]GTPγS labeled Gα subunits accumulate and can be measured by counting the amount of [35

Agonism: efficacy and potency

Agonists may be potent or weak irrespective of their efficacy. The [35S]GTPγS binding assay allows for construction of concentration-effect curves and therefore potency (EC50) and relative efficacy (Emax) measures. Thus, parameters measured are not subject to the amplification or other controls that have to be considered when measuring downstream systems, such as adenylyl cyclase, phospholipase C or gene expression, and so provide the best source of information about ligand-induced events at

Antagonism: characterization of receptors and agonist specificity

The stimulation of [35S]GTPγS binding by agonist gives a typical sigmoidal concentration effect curve and so the affinity of competitive antagonists can be measured using the [35S]GTPγS assay in much the same way as conventional bioassays. Shifts in the agonist concentration-effect curve for [35S]GTPγS binding in the presence of increasing concentrations of antagonist are analyzed by the method of Schild (Arunlakshana and Schild, 1959) to provide antagonist affinity as a pA2 value, defined as

Constitutive activity and inverse agonism

Agonist unoccupied receptors are able to constitutively activate G protein and this can be measured by [35S]GTPγS binding. Several native receptors show such constitutive activity that has been determined with the [35S]GTPγS assay, including delta opioid receptors in NG108-15 cells (Szekeres and Traynor, 1997) or C6 cells (Neilan et al., 1999), mu opioid receptors in HEK cells (Burford et al., 2000) and GH3 cells (Liu et al., 2001), native and recombinant histamine H3 receptors (Rouleau et al.,

[35S]GTPγS binding in clinical samples

GPCRs have been implicated in many human disease states and G proteins may be altered in pathophysiological states. Consequently, [35S]GTPγS binding has been measured in human post-mortem samples (Gonzalez-Maeso et al., 2002). Canabinoid CB1 receptor stimulated [35S]GTPγS binding is increased in caudate nucleus, putamen, globus paladus and substantia nigra of post mortem human brain samples from patients affected with Parkinson's disease compared to controls (Lastres-Becker et al., 2001). GTP

Newer assay formats

Recently, methodologies for the binding of [35S]GTPγS have been developed where there is no need to separate bound and free radioactivity. These assays are performed using either scintillation proximity assay (SPA) beads (Amersham Life Sciences) or Flashplates (Perkin Elmer Life Sciences) in either 96 or 384 well plate formats to increase throughput and allow for automation. In the SPA format, membranes and [35S]GTPγS are incubated with beads containing scintillant and coated with wheat germ

Concluding remarks

The [35S]GTPγS binding assay has provided much important pharmacological information because it is a relatively simple functional assay of receptor-mediated G protein activation that gives a large signal-to-noise ratio and is less likely to be subjected to modulation or amplification than more distant measures of receptor activation. The use of newer techniques, including receptor-G protein fusion proteins and selective identification of Gα subunits with antisera has increased the power of this

Acknowledgements

We thank NIDA (DA 00254 and DA 04087) for support.

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