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G-protein-coupled receptor activation is generally analyzed under equilibrium conditions. However, real-life receptor functions are often dependent on very short, transient stimuli that may not allow the achievement of a steady state. This is particularly true for synaptic receptors such as the α(2A)-adrenergic receptor (α(2A)-AR). Therefore, we developed a fluorescence resonance energy transfer-based technology to study nonequilibrium α(2A)-AR function in living cells. To examine the effects of increasing concentrations of the endogenous agonist norepinephrine on the speed and extent of α(2A)-AR activation with very high temporal resolution, we took advantage of a fluorophore-containing α(2A)-AR sensor. The results indicated 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 EC(50) values under nonequilibrium conditions start at ~26 μM (at 40 ms) and show a 10-fold decrease until the steady state is achieved. To analyze the ability of norepinephrine to trigger a downstream intracellular response after α(2A)-AR stimulation, we monitored the kinetics and amplitude of G(i) activation in real time by using a fluorophore-containing G(i) sensor. The results show that both the efficacy and the potency of norepinephrine in inducing G(i) activation achieve a steady state more slowly, compared with receptor activation, and that the initial EC(50) value of ~100 nM decreases in an exponential way, reaching the minimal value of ~10 nM at equilibrium. Therefore, both the efficacy and the potency of norepinephrine increase ~10-fold over a few seconds of agonist stimulation, which illustrates that receptor and G-protein signaling and signal amplification are highly time-dependent phenomena.

G protein-coupled receptors (GPCRs) are the key elements of a highly regulated transduction machinery that generates different signaling outcomes to hormones and neurotransmitters. Until recently, it was assumed that diverse ligands of a given GPCR differ only in their ability to alter the balance between the OFF and the ON state of the receptor. However, it has now become evident that their activation mechanisms are more complex and that receptors presumably display distinguishable active conformational states, which are induced by different agonists and correlate to specific signaling outputs. The use of different labeling strategies to insert fluorescent labels into purified, reconstituted receptors, or into receptors in intact cells, has made it possible to sense receptor activation via changes in their fluorescence. Here, we summarize recent progress in the analysis of agonist-dependent activation mechanisms of GPCRs acquired using modern spectroscopic and crystallographic techniques.

The concept of "functional selectivity" or "biased signaling" suggests that a ligand can have distinct efficacies with regard to different signaling pathways. We have investigated the question of whether biased signaling may be related to distinct agonist-induced conformational changes in receptors using the β(2)-adrenergic receptor (β(2)AR) and its two endogenous ligands epinephrine and norepinephrine as a model system. Agonist-induced conformational changes were determined in a fluorescently tagged β(2)AR FRET sensor. In this β(2)AR sensor, norepinephrine caused signals that amounted to only ≈50% of those induced by epinephrine and the standard "full" agonist isoproterenol. Furthermore, norepinephrine-induced changes in the β(2)AR FRET sensor were slower than those induced by epinephrine (rate constants, 47 versus 128 ms). A similar partial β(2)AR activation signal was revealed for the synthetic agonists fenoterol and terbutaline. However, norepinephrine was almost as efficient as epinephrine (and isoproterenol) in causing activation of G(s) and adenylyl cyclase. In contrast, fenoterol was quite efficient in triggering β-arrestin2 recruitment to the cell surface and its interaction with β(2)AR, as well as internalization of the receptors, whereas norepinephrine caused partial and slow changes in these assays. We conclude that partial agonism of norepinephrine at the β(2)AR is related to the induction of a different active conformation and that this conformation is efficient in signaling to G(s) and less efficient in signaling to β-arrestin2. These observations extend the concept of biased signaling to the endogenous agonists of the β(2)AR and link it to distinct conformational changes in the receptor.

The intrahelical salt bridge between \(E/D^{3.49}\) and \(R^{3.50}\) within the E/DRY motif on helix 3 (H3) and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of \(E^{3.49/6.30}\) in the thromboxane prostanoid (TP) receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA) with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET) indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow "resistant" to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes.

The development of several fluorescent approaches has enabled the monitoring the kinetics of GPCR activation and signalling in real-time. Such fluorescent-based assays—first established for isolated reconstituted receptors and later developed to follow the activation of GPCRs in living cells—suggest that GPCR activation can occur faster than previously thought and that different agonists may induce distinct conformations with different speeds of GPCRs activation. Some of those approaches have allowed the detection of both the modulation triggered by allosteric ligands on the receptor conformation and the transconformational switches occurring between two protomers constituting a heterodimer in intact cells. This chapter describes and summarizes recent studies on the agonist-dependent switching behaviour of GPCRs.

The structure-based design of a mutant form of the thromboxane A 2 prostanoid receptor (TP) was instrumental in characterizing the structural determinants of the hetero-dimerization process of this G protein coupled receptor (GPCR). The results suggest that the heterodimeric complexes between the TPα and β isoforms are characterized by contacts between hydrophobic residues in helix 1 from both monomers. Functional characterization confirms that TPα-TPβ hetero-dimerization serves to regulate TPα function through agonist-induced internalization, with important implications in cardiovascular homeostasis. The integrated approach employed in this study can be adopted to gain structural and functional insights into the dimerization/oligomerization process of all GPCRs for which the structural model of the monomer can be achieved at reasonable atomic resolution. © Springer Basel AG 2010.

In class A GPCRs the E/DRY motif is critical for receptor activation and function. According to experimental and computational data, R3.50 forms a double salt bridge with the adjacent E/D3.49 and E/D6.30 in helix 6, constraining the receptor in an inactive state. The disruption of this network of interactions facilitates conformational transitions that generate a signal or constitutive activity. Here we demonstrate that non-conservative substitution of either E129(3.49) or E240(6.30) of thromboxane prostanoid receptor (TP) resulted in mutants characterized by agonist-induced more efficient signaling properties, regardless of the G protein coupling. Results of computational modeling suggested a more effective interaction between G q and the agonist-bound forms of the TP mutants, compared to the wild type. Yet, none of the mutants examined revealed any increase in basal activity, precluding their classification as constitutively active mutants. Here, we propose that these alternative active conformations might be identified as superactive mutants or SAM. © 2010 Springer Basel AG.

Time-resolved fluorescence resonance energy transfer (TR-FRET) measurements with selective, fluorescently labeled ligands support the existence of GPCR dimers in native tissues and suggest that activated GPCR dimers are asymmetric.