Insights into GPCR pharmacology from the measurement of changes in intracellular cyclic AMP; advantages and pitfalls of differing methodologies

Abstract

It is clear that the G protein-coupled receptor family play a key role in the pharmaceutical industry, with a significant proportion of approved drugs targeting this protein class. While our growing understanding of the complexity of G protein-coupled receptor pharmacology is playing a key role in the future success of these endeavours, with allosteric mechanisms now well integrated into the industrial community and G protein-independent signalling mechanisms establishing themselves as novel phenomenon to be exploited, it is still possible to underestimate the complexity of G protein signal transduction mechanisms and the impact that inappropriate study of these mechanisms can have on data interpretation. In this manuscript we review different approaches to measuring the cAMP signal transduction pathway, with particular emphasis on key parameters influencing the data quality and biological relevance.

Figure 1

Influence of cAMP standard curves on the relationship between agonist concentration and measured response (cAMP or luminescence activity). (A) A cAMP standard curve determined for a competitive immunoassay-based enzyme complementation assay for cAMP (HitHunter, DiscoveRx). In this assay, cAMP competes for antibody binding against a cAMP analogue that is conjugated to a small peptide fragment of β-galactosidase. In the absence of free cAMP, the majority of this galactosidase conjugate of cAMP is captured by the antibody and is therefore unavailable for complementation to a larger fragment of β-galactosidase resulting in a low signal output. In the presence of free cAMP, antibody sites are occupied, leaving the cAMP conjugate free to complement with the large fragment of β-galactosidase, forming an active β-galactosidase enzyme that leads to substrate hydrolysis and the production of a chemiluminescent signal. Data points (Dr J.G. Baker, unpubl. data) are the mean of 6 replicates at each concentration of cAMP. (B) Simulated concentration–response curves for two agonists with a log EC₅₀ of −8.0 and maximal responses that differ by one order of magnitude (i.e. generating cAMP levels in each well of 400 and 4000 nM). (C) Resultant concentration–response curves generated from the concentration–response curves in (B) when converted to luminescence activity via interpolation from the cAMP standard curve in (A). Log EC₅₀ values obtained in (C) were −8.9 and −9.8 for agonists 2 and 1 respectively.

Figure 2

EPAC-based cAMP FRET sensor. (A) A schematic showing the basic design of an EPAC-based cAMP FRET sensor. In the absence of cAMP the conformation of the sensor is such that the fluorophores are in close proximity, generating FRET. Upon cAMP binding, conformational changes result in a decrease in FRET. (B) Top panel: Fluorescent images of HEK293T cells transiently transfected with a CFP/YFP FRET sensor containing the EPAC2 cAMP binding domain. CFP and YFP fluorescence are shown in blue and yellow respectively. Lower panel: A single cell trace plotted as a ratio of CFP: YFP intensity from HEK293T cells transiently tranfected with the EPAC2-based cAMP sensor. Exposure of cells to 5′-N-ethyl carboxamide adenosine at 30 s stimulates cAMP accumulation through an endogenously expressed adenosine A_2B receptor. Inhibition of cAMP accumulation results from the addition of the adenosine receptor antagonist xanthine amine congener at 300 s. CFP, cyan fluorescent protein; EPAC, exchange protein directly activated by cAMP; FRET, fluorescence resonance energy transfer; PDE, phosphodiesterase; YFP, yellow fluorescent protein.

Figure 3

Effect of the phosphodiesterase (PDE) inhibitor rolipram on ³H-cAMP breakdown and steady-state levels of cAMP in ³H-adenine-labelled brain slices from guinea-pig cerebral cortical slices. Data are taken from Donaldson et al. (1988a). In (A) brain slices were pre-incubated with 0.1 mM adenosine for 10 min to achieve a steady-state level. At time zero adenosine deaminase was added (1.2 U·mL⁻¹) to rapidly remove adenosine and cAMP levels then rapidly fell under the influence of endogenous PDEs. In (B) the same experiment was undertaken in the presence of the PDE inhibitor rolipram (0.1 mM). In this case a new and much higher steady-state level of cAMP was achieved. This is because the reduced PDE activity caused by rolipram allows the cAMP levels to rise until they achieve a new equilibrium at which cAMP synthesis is matched again by cAMP metabolism. The continued ability to metabolize cAMP under these conditions is clearly evident when adenosine deaminase is once again applied (B). The influence of the competitive PDE inhibitor rolipram is evident, however, in the reduced rate at which cAMP levels fall in (B). In both (A) and (B) the solid symbols show that the steady-state level of cAMP is well maintained in the absence of adenosine deaminase.