PT  - JOURNAL ARTICLE
AU  - Manuela Ambrosio
AU  - Martin J. Lohse
TI  - Non-equilibrium Activation of a G-protein-coupled Receptor
AID  - 10.1124/mol.112.077693
DP  - 2012 Jan 01
TA  - Molecular Pharmacology
PG  - mol.112.077693
4099  - http://molpharm.aspetjournals.org/content/early/2012/02/29/mol.112.077693.short
4100  - http://molpharm.aspetjournals.org/content/early/2012/02/29/mol.112.077693.full
AB  - G-protein coupled receptor activation is generally analyzed under equilibrium conditions. However, real-life receptor functions are often dependent on very short and transient stimuli that may not allow the achievement of the steady state. This is particularly true for synaptic receptors like the α2A-adrenergic receptor (AR). Therefore, we recently developed a fluorescence resonance energy transfer based technology to study non-equilibrium α2A-AR function in living cells. To examine the effects of increasing concentrations of the endogenous agonist norepinephrine on the speed and extent of α2A-AR activation with very high temporal resolution, we took advantage of an α2A-ARFlAsH/CFP sensor. The results indicate that the efficacy of norepinephrine in eliciting receptor activation increased in a time-dependent way reaching the maximum with a half-life of ≈60 ms. The EC50-values under non-equilibrium conditions start at ≈26 μM (t=40 ms) and show a 10-fold decrease until the steady state is achieved. To analyze norepinephrine ability in triggering a downstream intracellular response after α2A-AR stimulation, we monitored the kinetics and amplitude of Gi activation in real-time using a GiCFP/YFP sensor. The results show that both the efficacy and the potency of norepinephrine in inducing Gi activation achieve a steady state slower compared to receptor activation, and that the initial EC50-value of ≈100 nM decreases in an exponential way reaching the minimal value ≈10 nM at equilibrium. Thus, both efficacy and potency of norepinephrine increased about 10-fold over a few seconds of agonist stimulation, illustrating that receptor and G-protein signaling as well as signal amplification are highly time-dependent phenomena.