It is becoming increasingly clear that signaling via G proteincoupled receptors is a diverse phenomenon involving receptor interaction with a variety of signaling partners. Despite this diversity, receptor ligands are commonly classified only according to their ability to modify G protein-dependent signaling. Here we show that ␤2AR ligands like ICI118551 and propranolol, which are inverse agonists for Gs-stimulated adenylyl cyclase, induce partial agonist responses for the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) 1͞2 thus behaving as dual efficacy ligands. ERK1͞2 activation by dual efficacy ligands was not affected by ADP-ribosylation of G␣i and could be observed in S49-cyc ؊ cells lacking G␣s indicating that, unlike the conventional agonist isoproterenol, these drugs induce ERK1͞2 activation in a Gs͞i-independent manner. In contrast, this activation was inhibited by a dominant negative mutant of ␤-arrestin and was abolished in mouse embryonic fibroblasts lacking ␤-arrestin 1 and 2. The role of ␤-arrestin was further confirmed by showing that transfection of ␤-arrestin 2 in these knockout cells restored ICI118551 promoted ERK1͞2 activation. ICI118551 and propranolol also promoted ␤-arrestin recruitment to the receptor. Taken together, these observations suggest that ␤-arrestin recruitment is not an exclusive property of agonists, and that ligands classically classified as inverse agonists rely exclusively on ␤-arrestin for their positive signaling activity. This phenomenon is not unique to ␤2-adrenergic ligands because SR121463B, an inverse agonist on the V2 vasopressin receptor-stimulated adenylyl cyclase, recruited ␤-arrestin and stimulated ERK1͞2. These results point to a multistate model of receptor activation in which ligand-specific conformations are capable of differentially activating distinct signaling partners.

Current antidepressants are clinically effective only after several weeks of administration. Here, we show that serotonin(4) (5-HT(4)) agonists reduce immobility in the forced swimming test, displaying an antidepressant potential. Moreover, a 3 day regimen with such compounds modifies rat brain parameters considered to be key markers of antidepressant action, but that are observed only after 2-3 week treatments with classical molecules: desensitization of 5-HT(1A) autoreceptors, increased tonus on hippocampal postsynaptic 5-HT(1A) receptors, and enhanced phosphorylation of the CREB protein and neurogenesis in the hippocampus. In contrast, a 3 day treatment with the SSRI citalopram remains devoid of any effect on these parameters. Finally, a 3 day regimen with the 5-HT(4) agonist RS 67333 was sufficient to reduce both the hyperlocomotion induced by olfactory bulbectomy and the diminution of sucrose intake consecutive to a chronic mild stress. These findings point out 5-HT(4) receptor agonists as a putative class of antidepressants with a rapid onset of action.

G protein–coupled receptors (GPCRs) are important therapeutic targets that exhibit functional selectivity (biased signaling), in which different ligands or receptor variants elicit distinct downstream signaling. Understanding all the signaling events and biases that contribute to both the beneficial and adverse effects of GPCR stimulation by given ligands is important for drug discovery. Here, we report the design, validation, and use of pathway-selective bioluminescence resonance energy transfer (BRET) biosensors that monitor the engagement and activation of signaling effectors downstream of G proteins, including protein kinase C (PKC), phospholipase C (PLC), p63RhoGEF, and Rho. Combined with G protein and β-arrestin BRET biosensors, our sensors enabled real-time monitoring of GPCR signaling at different levels in downstream pathways in both native and engineered cells. Profiling of the responses to 14 angiotensin II (AngII) type 1 receptor (AT1R) ligands enabled the clustering of compounds into different subfamilies of biased ligands and showed that, in addition to the previously reported functional selectivity between Gαq and β-arrestin, there are also biases among G protein subtypes. We also demonstrated that biases observed at the receptor and G protein levels propagated to downstream signaling pathways and that these biases could occur through the engagement of different G proteins to activate a common effector. We also used these tools to determine how naturally occurring AT1R variants affected signaling bias. This suite of BRET biosensors provides a useful resource for fingerprinting biased ligands and mutant receptors and for dissecting functional selectivity at various levels of GPCR signaling.

A very important element controlling serotonin (5-HT) release throughout the brain is the 5-HT1A autoreceptor present on the soma and dendrites of 5-HT neurons since it exerts a negative feedback influence on their firing activity. This 5-HT1A autoreceptor receives an increased activation by endogenous 5-HT at the beginning of a treatment with a selective 5-HT reuptake inhibitor (SSRI) and, consequently, a decreased 5-HT neuronal firing activity is obtained. As the SSRI treatment is prolonged, the 5-HT1A autoreceptor desensitizes and firing activity is restored in the presence of the SSRI. That this adaptive change underlies, at least in part, the delayed therapeutic effect of SSRI in major depression is supported by the acceleration of the antidepressant response by the concomitant administration of the 5-HT1A autoreceptor antagonist pindolol with SSRIs.