Rate of [35S]GTP?S dissociation from g proteins reflects agonist efficacy

Kathleen Quirk & Philip G Strange, University of Reading, Whiteknights, Reading, RG4 7NH, UK.

The ability of agonists to stimulate the binding of GTP to G-proteins can be assessed by measuring the association of [35S]GTPγS. This assay has been used as a measure of agonist efficacy for many GPCRs. The amount of [35S]GTPγS bound provides a measure of the efficacy and the EC50 provides a measure of the potency of the compound. It was previously thought that the binding of [35S]GTPγS to the Gα subunit was irreversible (Sternweis & Robishaw, 1984). However, more recent studies have shown that [35S]GTPγS can be dissociated from the G-protein, and that its dissociation is accelerated in the presence of agonist (Traynor et. al., 2002). In this study we have taken the dopamine D2 receptor stably expressed in CHO cells as a system to investigate the dissociation of [35S]GTPγS binding. We examined a range of agonists of varying efficacy to stimulate dissociation of the pre-bound [35S]GTPγS from the Gα subunits.

[35S]GTPγS dissociation assays were carried out in triplicate in 20 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 0.1 mM DTT, pH 7.4 in a final volume of 1 ml. Each tube contained agonist at maximal effective concentration, 10 μM GDP, 0.1 nM [ 35S]GTPγS and 25 μg CHO cell membrane protein expressing the dopamine D2 receptor. Assays were incubated at 30oC for 60 min before addition of 10 μM GTPγS to initiate dissociation. Assays were then terminated by rapid filtration through Whatman GF/C filters using a Brandel 24 well cell harvester with 4 x 4 ml washes with ice cold phosphate buffered saline. Filters were soaked for a minimum of 6 hours in 2 ml Ultima Gold scintillation fluid before liquid scintillation counting. Data were fitted using GraphPad Prism (Graphpad San Diego, USA).

Dissociation data were best fitted to a model of a one site exponential decay. The full agonists dopamine and NPA (R(-)-propylnorapomorphine hydrochloride) resulted in the fastest dissociation of [35S]GTPγS, with rate constants of 0.28 ± 0.01 and 0.32 ± 0.02 min-1, respectively. The rate constants for the dissociation of [35S]GTPγS decreased for the partial agonists, with quinpirole (0.27 ± 0.06 min-1) > m-tyramine (0.19 ± 0.06 min-1) > bromocriptine (0.14 ± 0.02 min-1) > (+)-3-PPP (0.11 ± 0.01 min-1) = p-tyramine (0.10 ± 0.01 min-1). When these dissociation rate constants were plotted against the relative efficacy of the compounds a good correlation was seen with an r2 value of 0.92. The efficacy of each agonist was determined as the [35S]GTPγS bound in each experiment relative to that of 1mM dopamine at the start of the dissociation phase.

In conclusion we have shown that the binding of [35S]GTPγS to G-proteins is a reversible process in which binding can be dissociated. This dissociation is accelerated in the presence of agonists and the rate at which each agonist accelerates the rate of dissociation of [35S]GTPγS is determined by the efficacy of the agonist.

Sternweis P.C.& Robishaw J.D. (1984) J. Biol. Chem. 259 13806-13813.
Traynor J.R.et al. (2002) J. Pharmacol. Exp. Ther. 300 157-161.