Seven-transmembrane receptors, which constitute the largest, most ubiquitous and most versatile family of membrane receptors, are also the most common target of therapeutic drugs. Recent findings indicate that the classical models of G-protein coupling and activation of second-messenger-generating enzymes do not fully explain their remarkably diverse biological actions.

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...