Introduction
Receptors are kept in an inactive state until the binding of a ligand 'agonist' switches them into an active state within milliseconds1, 2. The transition between the inactive and active states involves the release of at least two important molecular constraints, known as the "ionic lock" between charged residues at the cytosolic sides of the receptor and the "rotamer toggle switch" in helix 6 (refs. 3–5). In this issue6, Yao and colleagues report a fluorescence method for following the breaking of the ionic lock in a GPCR, the 
2-adrenergic receptor (2AR), and identify agonists that differentially affect the two molecular switches. The new report represents an exciting development for dissecting molecular mechanisms that convert binding of ligands to activation of GPCRs.
GPCRs consist of seven membrane-spanning 
-helical structures (Fig. 1) and constitute the main family of cell surface receptors for both chemical stimuli (for example, hormones, neurotransmitters and chemoattractants) and sensory stimuli (for example, light, odorants and taste molecules). Binding of these extracellular 'agonist' ligands triggers receptor activation that couples to and activates signaling proteins such as heterotrimeric GTP-binding proteins (G proteins), which in turn modulate the flow of secondary messengers, such as cAMP, involved in critically important physiological processes (such as heartbeat). Key initiators of multiple biochemical signaling pathways, GPCRs are also involved in many pathologic processes and are targets of many of the available medical drugs used in humans7. Determining the molecular mechanisms by which this important family of receptors function is thus of paramount importance and could provide a molecular basis for the development of new therapies for many physiological disorders.
Figure 1: Molecular representation of a GPCR based on the X-ray crystal structure of rhodopsin (ref. 13).
Helix 6 is shown in green and helix 3 in blue. Figure prepared and kindly provided by Carsten Hoffmann (University of Würzburg, Germany).
Katie Ris
Full size image (42 KB)The 2-adrenergic receptor, which transmits signals for small neurotransmitters such as adrenaline, is a well-characterized receptor that serves as a model for dissecting mechanisms of activation of rhodopsin-like class A GPCRs. The keystone in the overall process of activating these receptors is a structural rearrangement of several transmembrane helices, in particular helices 3 and 6 (Fig. 1), triggered by presumably conserved switches that involve the disruption of an ionic interaction between the cytoplasmic face of helix 3 and helix 6 (ionic lock) and a rotamer toggle switch (modulation of the helix conformation around a conserved proline-kink) in helix 6 (refs. 8,9). These transmembrane movements expose receptor epitopes at the cytosolic side that should drive heterotrimeric G protein signaling. Ligands that bind to GPCRs may show different efficacies in activating a specific receptor, thereby eliciting full or partial receptor responses. The way that receptors differentiate full from partial agonists is still unclear. Two mechanisms can be envisioned: (i) partial agonists are 'partial' because they stabilize the active receptor conformation less effectively, thereby producing a smaller receptor response; or (ii) partial and full agonists switch the receptor into different conformations that have distinct activities. Previous studies support this latter model10, 11, and one attractive hypothesis is that the effectiveness of agonist action could arise from the ability to differentiate the ionic lock versus the rotamer toggle switch.
Yao et al.6 examined this hypothesis by probing the ability of a series of 2AR-agonists of varying efficacies (full and partial agonists) to trigger these two conformational switches involved in receptor activation. For these experiments, they used a purified mutant 2AR labeled with bright fluorophores to follow the ionic lock or the rotamer toggle switches. Disruption of the ionic lock can be monitored by the ability of a tryptophan residue close to the DRY motif at the intracellular side of helix 3 to quench the fluorescence of bimane covalently attached at the intracellular part of helix 6 of the receptor. In absence of agonist binding, the ionic lock keeps the receptor in an inactive state and separates the bimane-attached to helix 3 and the tryptophan in helix 6, preventing quenching of bimane fluorescence. Once a specific binding of agonist occurs, the receptor undergoes a conformational change that places bimane and tryptophan in close proximity. This event, detected by quenching of bimane fluorescence, is compatible with the breaking of the ionic lock between helix 3 and helix 6. In earlier studies of 2AR activation, the same authors used a receptor probe such that the change of fluorescence of tetramethylrhodamine attached to the third intracellular loop and close to helix 6 monitored a rotamer toggle switch12. Using these fluorescence approaches, Yao et al. highlight two remarkable features of the mechanisms of GPCR activation. First, partial agonists (such as dopamine) are effective as full agonists (such as epinephrine) in fully breaking the ionic lock and triggering the rotamer toggle switch. The action of full agonists must therefore be triggered by additional as-yet-unidentified switches in the receptor. Second, weak partial agonists such as salbutamol and catechol can have different effects on the two receptor switches, suggesting that the disruption of the ionic lock and the rotamer toggle switch in helix 6 might be independent molecular triggers. This raises the possibility that a specific molecule, by turning 'on' a particular receptor switch, might have the option to select a specific signaling pathway.
