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Optimising a [35S]GTPγS assay for agonist potency rather than signal-to-noise ratio In previous studies using standard [35S]GTP g S assay conditions optimised for screening (signal-to-noise ratio of 7:1) GRO-α stimulated [35S]GTPγ S binding, to CHO-CXCR2 membranes, produced a pEC50 of 7.02 ± 0.09 (n = 5). This was significantly lower than the pKi for GRO-α (10.15 ± 0.21, n = 4) obtained from radioligand binding studies (p<0.001, t-test). As it is known that the concentrations of various constituents of the [35S]GTPγ S assay can have an impact upon observed agonist potencies (Roberts et al, 2004; Siehler & Hoyer, 1999), we have examined the effect of varying [35S]GTP g S, GDP and Na+ concentration on pEC50, whilst monitoring the signal-to-noise ratio and assay window to potentially identify more appropriate assay conditions. CHO membranes (5 μg/well) stably expressing the human CXCR2 receptor were incubated with a range of concentrations of GRO-α (3 μM to 150 pM) and GDP (10, 2.5, 0.5 and 0.1 μM) for 10 minutes in assay buffer (20 mM HEPES, 10 mM MgCl2, 1 mM EDTA, 10 μg/mL saponin and 0.1 % (w/v) BSA) containing either 50 or 100 mM NaCl and 0.5 mg/well SPA beads. Various concentrations of [35S]GTPγ S (2, 1, 0.5, 0.25 and 0.1 nM – final assay concentration) were then added to a final well volume of 250 μL and further incubated for 60 minutes prior to centrifugation and detection of [35S]GTPγ S incorporation using a Topcount. Table 1
Table 1: Effect of varying [GDP], [35SGTPγS] and [Na+] on GRO- a pEC50, assay window and signal to noise ratio. Data expressed as mean ± s.e.m (n = 3) * indicates mean ± sd (n = 2). … indicates statistically different to that obtained with 0.1 μM GDP, ** indicates statistically different to that obtained with 0.1 nM [35S]GTPγS (p < 0.05; one-way ANOVA followed by Dunnett’s multiple comparison test) Table 1 shows that decreasing [GDP] leads to increases in apparent agonist potency. Although there was a concomitant decrease in signal-to-noise ratio, the assay window was largely unaffected. Increasing [35S]GTPγ S concentration increased both agonist potency and assay window but decreased signal-to-noise ratio. Significant improvements in signal-to-noise and assay window were seen with higher [Na+], but agonist potency was reduced. Interestingly, despite achieving a 10-fold increase in apparent GRO-α potency by modifying assay conditions, this was still 100-fold lower than its Ki at the CXCR2 receptor, also observed by Hall et al, (1999). In conclusion, although [35S]GTPγ S assays are often optimised for high signal-to-noise ratio, this results in lower observed agonist potencies. We have demonstrated that it is possible to optimise for agonist potency whilst retaining a suitable window size (cpm) and therefore suggest, that pEC50 may be a more important consideration when optimising conditions for agonist stimulated [35S]GTPγ S binding.
Roberts, D.J. et al., (2004) Mol. Pharmacol. 66, 1573-1579. |
