Many proteins associated with the plasma membrane are known to partition into submicroscopic sphingolipid- and cholesterol-rich domains called lipid rafts, but the determinants dictating this segregation of proteins in the membrane are poorly understood. We suppressed the tendency of Aequorea fluorescent proteins to dimerize and targeted these variants to the plasma membrane using several different types of lipid anchors. Fluorescence resonance energy transfer measurements in living cells revealed that acyl but not prenyl modifications promote clustering in lipid rafts. Thus the nature of the lipid anchor on a protein is sufficient to determine submicroscopic localization within the plasma membrane.

Signals transduced by kinases depend on the extent and duration of substrate phosphorylation. We generated genetically encoded fluorescent reporters for PKC activity that reversibly respond to stimuli activating PKC. Specifically, phosphorylation of the reporter expressed in mammalian cells causes changes in fluorescence resonance energy transfer (FRET), allowing real time imaging of phosphorylation resulting from PKC activation. Targeting of the reporter to the plasma membrane, where PKC is activated, reveals oscillatory phosphorylation in HeLa cells in response to histamine. Each oscillation in substrate phosphorylation follows a calcium oscillation with a lag of ∼10 s. Novel FRET-based reporters for PKC translocation, phosphoinositide bisphosphate conversion to IP3, and diacylglycerol show that in HeLa cells the oscillatory phosphorylations correlate with Ca2+-controlled translocation of conventional PKC to the membrane without oscillations of PLC activity or diacylglycerol. However, in MDCK cells stimulated with ATP, PLC and diacylglycerol fluctuate together with Ca2+ and phosphorylation. Thus, specificity of PKC signaling depends on the local second messenger-controlled equilibrium between kinase and phosphatase activities to result in strict calcium-controlled temporal regulation of substrate phosphorylation.

The concept of ligand bias at G protein-coupled receptors broadens the possibilities for agonist activities and provides the opportunity to develop safer, more selective therapeutics. Morphine pharmacology in b-arrestin-2 knockout mice suggested that a ligand that promotes coupling of the m-opioid receptor (MOR) to G proteins, but not b-arrestins, would result in higher analgesic efficacy, less gastrointestinal dysfunction, and less respiratory suppression than morphine. Here we report the discovery of TRV130 ([(3-methoxythiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethyl})amine), a novel MOR G protein-biased ligand. In cell-based assays, TRV130 elicits robust G protein signaling, with potency and efficacy similar to morphine, but with far less b-arrestin recruitment and receptor internalization. In mice and rats, TRV130 is potently analgesic while causing less gastrointestinal dysfunction and respiratory suppression than morphine at equianalgesic doses. TRV130 successfully translates evidence that analgesic and adverse MOR signaling pathways are distinct into a biased ligand with differentiated pharmacology. These preclinical data suggest that TRV130 may be a safer and more tolerable therapeutic for treating severe pain.

For many years, ␤-adrenergic receptor antagonists (␤-blockers or ␤AR antagonists) have provided significant morbidity and mortality benefits in patients who have sustained acute myocardial infarction. More recently, ␤-adrenergic receptor antagonists have been found to provide survival benefits in patients suffering from heart failure, although the efficacy of different ␤-blockers varies widely in this condition. One drug, carvedilol, a nonsubtypeselective ␤AR antagonist, has proven particularly effective in the treatment of heart failure, although the mechanism(s) responsible for this are controversial. Here, we report that among 16 clinically relevant ␤AR antagonists, carvedilol displays a unique profile of in vitro signaling characteristics. We observed that in ␤2 adrenergic receptor (␤2AR)-expressing HEK-293 cells, carvedilol has inverse efficacy for stimulating G s-dependent adenylyl cyclase but, nonetheless, stimulates (i) phosphorylation of the receptor's cytoplasmic tail on previously documented G protein-coupled receptor kinase sites; (ii) recruitment of ␤-arrestin to the ␤2AR; (iii) receptor internalization; and (iv) activation of extracellular regulated kinase 1/2 (ERK 1/2), which is maintained in the G protein-uncoupled mutant ␤2AR T68F,Y132G,Y219A (␤2AR TYY ) and abolished by ␤-arrestin2 siRNA. Taken together, these data indicate that carvedilol is able to stabilize a receptor conformation which, although uncoupled from Gs, is nonetheless able to stimulate ␤-arrestinmediated signaling. We hypothesize that such signaling may contribute to the special efficacy of carvedilol in the treatment of heart failure and may serve as a prototype for a new generation of therapeutic ␤2AR ligands.␤-adrenergic receptor ͉ antagonist ͉ ERK 1/2 ͉ scaffold ͉ internalization

Deleterious effects on the heart from chronic stimulation of β-adrenergic receptors (βARs), members of the 7 transmembrane receptor family, have classically been shown to result from G s -dependent adenylyl cyclase activation. Here, we identify a new signaling mechanism using both in vitro and in vivo systems whereby β-arrestins mediate β 1 AR signaling to the EGFR. This β-arrestin-dependent transactivation of the EGFR, which is independent of G protein activation, requires the G protein-coupled receptor kinases 5 and 6. In mice undergoing chronic sympathetic stimulation, this novel signaling pathway is shown to promote activation of cardioprotective pathways that counteract the effects of catecholamine toxicity. These findings suggest that drugs that act as classical antagonists for G protein signaling, but also stimulate signaling via β-arrestin-mediated cytoprotective pathways, would represent a novel class of agents that could be developed for multiple members of the 7 transmembrane receptor family.Introduction β-Adrenergic receptors (βARs) belong to the family of 7 transmembrane receptors (7TMRs) (1) and mediate the powerful regulatory effects on cardiac function of the catecholamine neurotransmitters epinephrine and norepinephrine. β 1 ARs constitute more than 70% of the cardiac βARs. Catecholamine stimulation of β 1 ARs results in activation of heterotrimeric G proteins followed by rapid phosphorylation of the receptor, resulting in desensitization (2). Homologous desensitization of β 1 ARs is brought about by phosphorylation of the receptor by G protein-coupled receptor kinases (GRKs), leading to the recruitment of β-arrestin, which then sterically interdicts further coupling to G proteins (3) and targets the receptor for internalization (3). In addition to β-arrestin's role in terminating G protein signaling, recent studies demonstrate that β-arrestins also function as adapter molecules, allowing for the assembly of multiprotein signaling complexes such as ERKs and tyrosine kinases (4, 5). For the angiotensin II type 1A receptor (AT 1A R), this second wave of β-arrestin-mediated signaling has recently been demonstrated to be independent of G protein signaling (6) and to require the activity of GRKs 5 and 6 (7).The signaling mechanisms that underlie the activation of the mitogenic ERK growth response by 7TMRs are complex and likely result from both classical G protein-regulated effectors such as PKA and PKC and non-G protein-mediated crosstalk, such as EGFR transactivation (8). The current paradigm of transactivation involves agonist stimulation of a 7TMR, which through a number of undefined steps leads to MMP-mediated cleavage and

No abstract

Seven transmembrane receptors (7TMRs), commonly referred to as G protein-coupled receptors, form a large part of the "druggable" genome. 7TMRs can signal through parallel pathways simultaneously, such as through heterotrimeric G proteins from different families, or, as more recently appreciated, through the multifunctional adapters, ␤-arrestins. Biased agonists, which signal with different efficacies to a receptor's multiple downstream pathways, are useful tools for deconvoluting this signaling complexity. These compounds may also be of therapeutic use because they have distinct functional and therapeutic profiles from "balanced agonists." Although some methods have been proposed to identify biased ligands, no comparison of these methods applied to the same set of data has been performed. Therefore, at this time, there are no generally accepted methods to quantify the relative bias of different ligands, making studies of biased signaling difficult. Here, we use complementary computational approaches for the quantification of ligand bias and demonstrate their application to two well known drug targets, the ␤2 adrenergic and angiotensin II type 1A receptors. The strategy outlined here allows a quantification of ligand bias and the identification of weakly biased compounds. This general method should aid in deciphering complex signaling pathways and may be useful for the development of novel biased therapeutic ligands as drugs.

Biased G protein-coupled receptor ligands engage subsets of the receptor signals normally stimulated by unbiased agonists. However, it is unclear whether ligand bias can elicit differentiated pharmacology in vivo. Here, we describe the discovery of a potent, selective ␤-arrestin biased ligand of the angiotensin II type 1 receptor. TRV120027 (Sar-Arg-Val-Tyr-Ile-His-Pro-DAla-OH) competitively antagonizes angiotensin II-stimulated G protein signaling, but stimulates ␤-arrestin recruitment and activates several kinase pathways, including p42/44 mitogenactivated protein kinase, Src, and endothelial nitric-oxide synthase phosphorylation via ␤-arrestin coupling. Consistent with ␤-arrestin efficacy, and unlike unbiased antagonists, TRV120027 increased cardiomyocyte contractility in vitro. In rats, TRV120027 reduced mean arterial pressure, as did the unbiased antagonists losartan and telmisartan. However, unlike the unbiased antagonists, which decreased cardiac performance, TRV120027 increased cardiac performance and preserved cardiac stroke volume. These striking differences in vivo between unbiased and ␤-arrestin biased ligands validate the use of biased ligands to selectively target specific receptor functions in drug discovery.