Multiple Switches in G Protein-Coupled Receptor Activation

The activation mechanism of G protein-coupled receptors has presented a puzzle that finally may be close to solution. These receptors have a relatively simple architecture consisting of seven transmembrane helices that contain just a handful of highly conserved amino acids, yet they respond to light and a range of chemically diverse ligands. Recent NMR structural studies on the active metarhodopsin II intermediate of the visual receptor rhodopsin, along with the recent crystal structure of the apoprotein opsin, have revealed multiple structural elements or ‘switches’ that must be simultaneously triggered to achieve full activation. The confluence of several required structural changes is an example of “coincidence counting”, which is often used by nature to regulate biological processes. In ligand-activated G protein-coupled receptors, the presence of multiple switches may provide an explanation for the differences between full, partial and inverse agonists.

Introduction

G protein-coupled receptors (GPCRs) are integral membrane proteins that respond to light (rhodopsin-like receptors) and a diverse array of chemical signals. Common features of these receptors are that they have seven transmembrane helices and activate intracellular G proteins. Despite the cellular and pharmacological importance of GPCRs, it has been a challenge to establish how the seven transmembrane helix structure recognizes and responds to such a large and diverse set of ligands. One of the first insights into how these receptors are activated came from site-directed spin labeling studies using electron paramagnetic resonance (EPR) spectroscopy on the visual pigment rhodopsin that revealed a large outward rotation of transmembrane helix H6 [1]. Crosslinking of helices H3 and H6 demonstrated that this motion was required for formation of the G-protein binding site on the intracellular surface of the receptor 1, 2. In 2000, the first of several crystal structures of rhodopsin 3, 4, 5 and its photoreaction intermediates 6, 7 was reported (Figure 1a). More recently, the crystal structures of several ligand-activated GPCRs have been solved using different methods to stabilize inactive receptor conformations 8, 9, 10. Because the transition to the active receptor appears to require a fluid membrane environment to accommodate an appreciable change in receptor structure, it has been a challenge to obtain high-resolution structures of active GPCRs.

In the past year, the landscape has shifted with the reported crystal structure of the apoprotein opsin 11, 12 and nuclear magnetic resonance (NMR) structural studies of the active metarhodopsin II intermediate (meta II) 13, 14 (Figure 2). The structural information that emerges from NMR and protein crystallography is extremely complementary. The NMR studies have focused on structural changes on the extracellular side of the receptor caused by retinal isomerization. The NMR data show that the second extracellular loop (EL2), which is wedged between two of the transmembrane helices (H3 and H6), is displaced upon activation (Figure 1b). The opsin structure lacks the agonist all-trans retinal chromophore on the extracellular side of the receptor, but retains elements of the active structure on the intracellular side of the receptor. Importantly, it captures the outward rotation of H6 and demonstrates that this creates the binding site for the C terminus of the Gα subunit of transducin (Figure 1c). The picture that emerges is that EL2 is a regulatory element that prevents the outward rotation of the intracellular end of H6, perhaps by preventing the inward pivoting of the extracellular end of H6 as envisioned by the global toggle switch mechanism [15].

This simple “clothes-pin” model is not adequate to fully describe the activation mechanism. In addition, the intracellular ends of helices H3 and H6 are clamped together by an ionic lock between Arg135^3.50 on H3 and Glu247^6.30 on H6 16, 17. What has not been clear until recently is how this lock is released. The combination of NMR and X-ray crystallography provides an answer. Retinal isomerization displaces EL2 from the retinal-binding site. The motion of EL2 is tightly coupled to the reorientation of helices H5, H6 and H7. On the intracellular side of H5 and H7 are two conserved tyrosines, Tyr223^5.58 on H5 and Tyr306^7.53 on H7. The coordinated motion of H5-H7 places these tyrosines between the intracellular ends of helices H3 and H6 disrupting the ionic lock 11, 12.

In this review, we first describe the multiple structural “switches” in rhodopsin that trigger the conformational changes involved in activation and formation of the G-protein binding site. We then draw parallels with ligand-activated GPCRs, for which the occurrence of multiple switches may explain the structural differences between full, partial and inverse agonists 18, 19. The concept of molecular switches extends the concept of structural motifs as functional microdomains developed by Weinstein and colleagues [20] who identified several conserved motifs in GPCRs that mediate receptor function.