β-Arrestin-biased Agonism at the β2-Adrenergic Receptor

Abstract

Classically, the ␤ 2 -adrenergic receptor (␤ 2 AR) and other members of the seven-transmembrane receptor (7TMR) superfamily activate G protein-dependent signaling pathways in response to ligand stimulus. It has recently been discovered, however, that a number of 7TMRs, including ␤ 2 AR, can signal via ␤-arrestin-dependent pathways independent of G protein activation. It is currently unclear if among ␤ 2 AR agonists there exist ligands that disproportionately signal via G proteins or ␤-arrestins and are hence "biased." Using a variety of approaches that include highly sensitive fluorescence resonance energy transfer-based methodologies, including a novel assay for receptor internalization, we show that the majority of known ␤ 2 AR agonists exhibit relative efficacies for ␤-arrestin-associated activities (␤-arrestin membrane translocation and ␤ 2 AR internalization) identical to their relative efficacies for G proteindependent signaling (cyclic AMP generation). However, for three ␤AR ligands there is a marked bias toward ␤-arrestin signaling; these ligands stimulate ␤-arrestin-dependent receptor activities to a much greater extent than would be expected given their efficacy for G protein-dependent activity. Structural comparison of these biased ligands reveals that all three are catecholamines containing an ethyl substitution on the ␣-carbon, a motif absent on all of the other, unbiased ligands tested. Thus, these studies demonstrate the potential for developing a novel class of 7TMR ligands with a distinct bias for ␤-arrestin-mediated signaling.7TMRs, 5 also known as G protein-coupled receptors, are the largest family of proteins involved in the transduction of signals from the extracellular milieu to intracellular effectors (1) and account for nearly 2% of all human genes (2). Clinically, 7TMR ligands are the single largest class of pharmacologic agents (3). According to a well established and evolutionarily conserved paradigm, 7TMRs signal through direct activation of heterotrimeric G proteins to promote the activation or inhibition of second messenger-generating enzymes and changes in second messenger-dependent effector activities (4). Thus, for example, agonist stimulation of ␤ 2 AR promotes G s -mediated activation of adenylyl cyclase, with subsequent cAMP production and cAMP-associated signaling events. Signal termination and 7TMR desensitization result from recruitment of ␤-arrestin proteins to the cytoplasmic surface of 7TMR following agoniststimulated receptor phosphorylation by the G protein-coupled receptor kinase (GRK) family of proteins (5, 6).When originally identified, the ␤-arrestin proteins were believed to play a role only in limiting 7TMR signaling by physically interceding between the receptor and G protein. Recent work has demonstrated, however, that for a variety of 7TMRs, ␤-arrestin proteins can mediate G protein-independent 7TMR signaling by selectively scaffolding signaling cascade components, including small GTP-binding proteins and members of the MAPK cascade (5, 7). These findings have...

Coarse-grained modeling of allosteric regulation in protein receptors

“…Although that analysis needs to be performed at the atomic level, it may be limited to the orthosteric site environment, which is much smaller than the entire receptor and has much shorter relaxation times. As such, the proposed strategy could facilitate the design of receptor-targeting drugs with higher selectivity and reduced side effects (51)(52)(53). Although a comprehensive atomic-scale theory of the receptor signaling response lies ahead, our results indicate that key features of the allosteric regulation mechanisms in receptors are revealed at a CG level.…”

Distinct conformational changes in β-arrestin report biased agonism at seven-transmembrane receptors

“…To examine whether phosphorylation-independent conformational changes in ␤-arrestin exist for other receptors, we next used a ␤2AR mutant that lacks both GRK and PKA phosphorylation sites (␤2AR GRK-/PKA-). This mutant receptor does not exhibit ␤-arrestin-mediated ERK signaling when tested at endogenous levels of ␤-arrestin (18), and ␤-arrestin recruitment to this mutant receptor could not be detected by confocal microscopy and cross-linking studies (18). However, a FRET-based approach revealed weak recruitment of ␤-arrestin to ␤2AR GRK-/PKA-upon high expression levels of exogenous ␤-arrestin (25).…”

“…To correlate the efficacy of a given ligand at its cognate receptor with the conformational change in ␤-arrestin, we investigated seven additional ligands for the ␤2AR (in addition to isoproterenol and propranolol) with varying efficacies in terms of cAMP response and ␤-arrestin recruitment (18) in this BRET assay. We monitored the changes in BRET ratio upon stimulation of both the wild-type ␤2AR and ␤2AR TYY .…”

Differential effects of opioid agonists on G protein expression in CHO cells expressing cloned human opioid receptors

“…As postulated by Kenakin (Kenakin, 2003;Kenakin, 2005), all seven transmembrane receptors, such as β2-adrenergic receptors (Drake et al, 2008), are likely capable of adopting a range of distinct conformations, each of which can lead to the activation of distinct intracellular signaling pathways. The adoption of these distinct conformations can, in turn, be modulated by the presence of ligand.…”