SUMMARY Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid β-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, β-arrestin, and G protein. These super-complexes or “megaplexes” more readily form at receptors that interact strongly with β-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with β-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.

The concepts of functional selectivity and ligand bias are becoming increasingly appreciated in modern drug discovery programs, necessitating more informed approaches to compound classification and, ultimately, therapeutic candidate selection. Using the b2-adrenergic receptor as a model, we present a proof of concept study that assessed the bias of 19 b-adrenergic ligands, including many clinically used compounds, across four pathways [cAMP production, extracellular signal-regulated kinase 1/2 (ERK1/2) activation, calcium mobilization, and receptor endocytosis] in the same cell background (human embryonic kidney 293S cells). Efficacy-based clustering placed the ligands into five distinct groups with respect to signaling signatures. In some cases, apparent functional selectivity originated from offtarget effects on other endogenously expressed adrenergic receptors, highlighting the importance of thoroughly assessing selectivity of the responses before concluding receptor-specific ligand-biased signaling. Eliminating the nonselective compounds did not change the clustering of the 10 remaining compounds. Some ligands exhibited large differences in potency for the different pathways, suggesting that the nature of the receptoreffector complexes influences the relative affinity of the compounds for specific receptor conformations. Calculation of relative effectiveness (within pathway) and bias factors (between pathways) for each of the compounds, using an operational model of agonism, revealed a global signaling signature for all of the compounds relative to isoproterenol. Most compounds were biased toward ERK1/2 activation over the other pathways, consistent with the notion that many proximal effectors converge on this pathway. Overall, we demonstrate a higher level of ligand texture than previously anticipated, opening perspectives for the establishment of pluridimensional correlations between signaling profiles, drug classification, therapeutic efficacy, and safety.

The recognition that individual GPCRs can activate multiple signaling pathways has raised the possibility of developing drugs selectively targeting therapeutically relevant ones. This requires tools to determine which G proteins and barrestins are activated by a given receptor. Here, we present a set of BRET sensors monitoring the activation of the 12 G protein subtypes based on the translocation of their effectors to the plasma membrane (EMTA). Unlike most of the existing detection systems, EMTA does not require modification of receptors or G proteins (except for Gs). EMTA was found to be suitable for the detection of constitutive activity, inverse agonism, biased signaling and polypharmacology. Profiling of 100 therapeutically relevant human GPCRs resulted in 1,500 pathway-specific concentration-response curves and revealed a great diversity of coupling profiles ranging from exquisite selectivity to broad promiscuity. Overall, this work describes unique resources for studying the complexities underlying GPCR signaling and pharmacology.

Classically, the prostaglandin E 2 (PGE 2 ) receptor EP 4 has been classified as coupling to the G␣ s subunit, leading to intracellular cAMP increases. However, EP 4 signaling has been revealed to be more complex and also involves coupling to pertussis toxinsensitive G␣ i proteins and ␤-arrestin-mediated effects. There are now many examples of selective activation of independent pathways by G protein-coupled receptor (GPCR) ligands, a concept referred to as functional selectivity. Because most EP 4 ligands had thus far only been functionally characterized by their ability to stimulate cAMP production, we systematically determined the potencies and efficacies of a panel of EP 4 ligands for activation of G␣ s , G␣ i , and ␤-arrestin relative to the endogenous ligand PGE 2 . For this purpose, we adapted three bioluminescence resonance energy transfer (BRET) assays to evaluate the respective pathways in living cells. Our results suggest considerable functional selectivity among the tested, structurally related agonists. PGE 2 was the most selective in activating G␣ s , whereas PGF 2␣ and PGE 1 alcohol were the most biased for activating G␣ i1 and ␤-arrestin, respectively. We observed reversal in order of potencies between ␤-arrestin 2 and G␣ i1 functional assays comparing PGE 1 alcohol and either PGF 2␣ , PGD 2 , or 7-[(1R,2R)-2-[(E,3R)-3-hydroxy-4-(phenoxy)-but-1-enyl]-5-oxocyclopentyl]heptanoic acid (M&B28767). Most ligands were full agonists for the three pathways tested. Our results have implications for the use of PGE 2 analogs in experimental and possibly clinical settings, because their activity spectra on EP 4 differ from that of the native agonist. The BRET-based methodology used for this first systematic assessment of a set of EP 4 agonists should be applicable for the study of other GPCRs.Prostanoids (prostaglandins and thromboxane) are lipid hormone mediators derived from cyclooxygenase-catalyzed metabolism of arachidonic acid. Prostaglandin E 2 (PGE 2 ) is the most widely produced prostanoid in the body. Its various effects include the contraction or relaxation of smooth muscle, modulation of immune responses, and the regulation of the production of a variety of cytokines. PGE 2 is also implicated in several pathologies including cancer, inflammation, and hypertension. Four G protein-coupled receptors (GPCRs) designated subtypes EP 1 through EP 4 , which activate different G protein-dependent signaling pathways but are often expressed in the same cell types, mediate the effects of PGE 2 . The biology of responses to PGE 2 is correspondingly complex, but evidence is increasing that EP 4 is a critical determinant for the role of PGE 2 in carcinogenesis and cancer progression (Fulton et al., 2006). Accordingly, studies using mice lacking the EP 4 receptor [EP 4 (Ϫ/Ϫ)] and EP 4 -selective ligands have revealed a role for this receptor in the progression of colon carcinogenesis, in