Physiological effects of ␤ adrenergic receptor (␤2AR) stimulation have been classically shown to result from G s -dependent adenylyl cyclase activation. Here we demonstrate a novel signaling mechanism wherein ␤-arrestins mediate ␤2AR signaling to extracellularsignal regulated kinases 1/2 (ERK 1/2) independent of G protein activation. Activation of ERK1/2 by the ␤2AR expressed in HEK-293 cells was resolved into two components dependent, respectively, on G s -G i /protein kinase A (PKA) or ␤-arrestins. G proteindependent activity was rapid, peaking within 2-5 min, was quite transient, was blocked by pertussis toxin (G i inhibitor) and H-89 (PKA inhibitor), and was insensitive to depletion of endogenous ␤-arrestins by siRNA. ␤-Arrestin-dependent activation was slower in onset (peak 5-10 min), less robust, but more sustained and showed little decrement over 30 min. It was insensitive to pertussis toxin and H-89 and sensitive to depletion of either ␤-arrestin1 or -2 by small interfering RNA. In G s knock-out mouse embryonic fibroblasts, wild-type ␤2AR recruited ␤-arrestin2-green fluorescent protein and activated pertussis toxin-insensitive ERK1/2. Furthermore, a novel ␤2AR mutant (␤2AR T68F,Y132G,Y219A or ␤2AR TYY ), rationally designed based on Evolutionary Trace analysis, was incapable of G protein activation but could recruit ␤-arrestins, undergo ␤-arrestin-dependent internalization, and activate ␤-arrestin-dependent ERK. Interestingly, overexpression of GRK5 or -6 increased mutant receptor phosphorylation and ␤-arrestin recruitment, led to the formation of stable receptor-␤-arrestin complexes on endosomes, and increased agonist-stimulated phospho-ERK1/2. In contrast, GRK2, membrane translocation of which requires G␤␥ release upon G protein activation, was ineffective unless it was constitutively targeted to the plasma membrane by a prenylation signal (CAAX). These findings demonstrate that the ␤2AR can signal to ERK via a GRK5/6-␤-arrestin-dependent pathway, which is independent of G protein coupling.The ␤2-adrenergic receptor (␤2AR) 4 is a well studied member of the large and diverse group of seven transmembrane receptors (7TMRs), which have been shown classically to exert their intracellular effects through G protein activation (1-3). Agonist stimulation of the ␤2AR leads to G s -mediated activation of adenylyl cyclase, resulting in the production of cAMP and subsequent downstream signaling events. Moreover, additional studies both in cultured cell lines and in vitro have demonstrated that, in response to agonist, the ␤2AR can undergo PKAdependent phosphorylation leading to activation of G i (a process referred to as G protein "switching"), thereby effectively changing the signaling specificity of the receptor (4).Cessation of agonist-activated ␤2AR-G s -mediated signaling occurs via recruitment of modulatory proteins, ␤-arrestins, to the cytoplasmic surface of the receptor, a process that is enhanced by receptor phosphorylation by G protein-coupled receptor kinases (GRKs) (5). ␤-arrestin binding physically pre...

The functions of G-protein coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein coupled receptor kinases (GRKs), and the arrestins1. G proteins mediate activation of second messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors2, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization3. Arrestins activated by interaction with phosphorylated receptors can also mediate G protein-independent signaling by serving as adaptors to link receptors to numerous signaling pathways4. Despite their central role in regulation and signaling of GPCRs, a structural understanding of β-arrestin activation and interaction with GPCRs is still lacking. Here, we report the crystal structure of β-arrestin1 in complex with a fully phosphorylated 29 amino acid carboxy-terminal peptide derived from the V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate β-arrestin15. To capture this active conformation, we utilized a conformationally-selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of β-arrestin1. The structure of the β-arrestin1:V2Rpp:Fab30 complex shows striking conformational differences in β-arrestin1 compared to its inactive conformation. These include rotation of the amino and carboxy-terminal domains relative to each other, and a major reorientation of the “lariat loop” implicated in maintaining the inactive state of β-arrestin1. These results reveal, for the first time at high resolution, a receptor-interacting interface on β-arrestin, and they suggest a potentially general molecular mechanism for activation of these multifunctional signaling and regulatory proteins.

Phosphorylation of G protein–coupled receptors (GPCRs, which are also known as seven-transmembrane spanning receptors) by GPCR kinases (GRKs) plays essential roles in the regulation of receptor function by promoting interactions of the receptors with β-arrestins. These multifunctional adaptor proteins desensitize GPCRs, by reducing receptor coupling to G proteins and facilitating receptor internalization, and mediate GPCR signaling through β-arrestin–specific pathways. Detailed mapping of the phosphorylation sites on GPCRs targeted by individual GRKs and an understanding of how these sites regulate the specific functional consequences of β-arrestin engagement may aid in the discovery of therapeutic agents targeting individual β-arrestin functions. The β2-adrenergic receptor (β2AR) has many serine and threonine residues in the carboxyl-terminal tail and the intracellular loops, which are potential sites of phosphorylation. We monitored the phosphorylation of the β2AR at specific sites upon stimulation with an agonist that promotes signaling by both G protein–mediated and β-arrestin–mediated pathways or with a biased ligand that promotes signaling only through β-arrestin–mediated events in the presence of the full complement of GRKs or when either GRK2 or GRK6 was depleted. We correlated the specific and distinct patterns of receptor phosphorylation by individual GRKs with the functions of β-arrestins and propose that the distinct phosphorylation patterns established by different GRKs establish a “barcode” that imparts distinct conformations to the recruited β-arrestin, thus regulating its functional activities.