Despite recent advances in crystallography of G protein-coupled receptors (GPCRs), little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here, we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously binding the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into activation mechanism and allosteric modulation of muscarinic receptors.

The concept of intrinsic efficacy has been enshrined in pharmacology for half of a century, yet recent data have revealed that many ligands can differentially activate signaling pathways mediated via a single G protein-coupled receptor in a manner that challenges the traditional definition of intrinsic efficacy. Some terms for this phenomenon include functional selectivity, agonist-directed trafficking, and biased agonism. At the extreme, functionally selective ligands may be both agonists and antagonists at different functions mediated by the same receptor. Data illustrating this phenomenon are presented from serotonin, opioid, dopamine, vasopressin, and adrenergic receptor systems. A variety of mechanisms may influence this apparently ubiquitous phenomenon. It may be initiated by differences in ligand-induced intermediate conformational states, as shown for the ␤ 2 -adrenergic receptor. Subsequent mechanisms that may play a role include diversity of G proteins, scaffolding and signaling partners, and receptor oligomers. Clearly, expanded research is needed to elucidate the proximal (e.g., how functionally selective ligands cause conformational changes that initiate differential signaling), intermediate (mechanisms that translate conformation changes into differential signaling), and distal mechanisms (differential effects on target tissue or organism). Besides the heuristically interesting nature of functional selectivity, there is a clear impact on drug discovery, because this mechanism raises the possibility of selecting or designing novel ligands that differentially activate only a subset of functions of a single receptor, thereby optimizing therapeutic action. It also may be timely to revise classic concepts in quantitative pharmacology and relevant pharmacological conventions to incorporate these new concepts. Receptor Pharmacology for the New MillenniumFor the last half-century, pharmacological theory has posited that ligands could be characterized by the nature of the functional effects elicited by their interaction with their tar-

The past decade has witnessed a significant growth in the identification of allosteric modulators of G protein-coupled receptors (GPCRs), i.e., ligands that interact with binding sites that are topographically distinct from the orthosteric site recognized by the receptor's endogenous agonist. Because of their ability to modulate receptor conformations in the presence of orthosteric ligand, allosteric modulators can "fine-tune" classical pharmacological responses. This is advantageous in terms of a potential for engendering greater GPCR subtype-selectivity, but represents a significant challenge for detecting and validating allosteric behaviors. Although allosteric sites need not have evolved to accommodate endogenous ligands, there are a number of examples of where such modulators have been shown to contribute to physiological or pathophysiological processes. Studies are also beginning to unravel the structural basis of allosteric modulation of GPCRs. It remains to be determined whether such modulation represents interactions within monomers versus across dimers.

SUMMARY Class B G protein-coupled receptors are major targets for treatment of chronic diseases, including osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric Gαβγs protein determined by Volta phase plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor through binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7. This conformation is accompanied by a 60° kink in helix 6 and large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gαs. Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G protein coupling via interaction with the Gβ subunit. This structure provides a new framework for understanding G protein-coupled receptor function.