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...

Primary cilia function as a sensory signaling compartment in processes ranging from mammalian Hedgehog signaling to neuronal control of obesity. Intraflagellar transport (IFT) is an ancient, conserved mechanism required to assemble cilia and for trafficking within cilia. The link between IFT, sensory signaling, and obesity is not clearly defined, but some novel monogenic obesity disorders may be linked to ciliary defects. The tubby mouse, which presents with adult-onset obesity, arises from mutation in the Tub gene. The tubby-like proteins comprise a related family of poorly understood proteins with roles in neural development and function. We find that specific Tubby family proteins, notably Tubby-like protein 3 (TULP3), bind to the IFT-A complex. IFT-A is linked to retrograde ciliary transport, but, surprisingly, we find that the IFT-A complex has a second role directing ciliary entry of TULP3. TULP3 and IFT-A, in turn, promote trafficking of a subset of G protein-coupled receptors (GPCRs), but not Smoothened, to cilia. Both IFT-A and membrane phosphoinositide-binding properties of TULP3 are required for ciliary GPCR localization. TULP3 and IFT-A proteins both negatively regulate Hedgehog signaling in the mouse embryo, and the TULP3–IFT-A interaction suggests how these proteins cooperate during neural tube patterning.

The type I PTH/PTH-related peptide receptor (PTH1R), 2 a seventransmembrane receptor (7TMR) highly expressed in the kidney and bone, plays a fundamental role in the regulation of calcium homeostasis, as well as in bone formation and resorption. Ligands for PTH1R including PTHrp and PTH are involved in the etiology and treatment of disease processes such as hypercalcemia of malignancy and osteoporosis. The actions of PTH, however, are complex. PTH is known for both anabolic and catabolic effects on bone, which are dependent upon intermittent or persistent exposure, respectively (1-3). The mechanistic basis of these effects on bone remodeling are not well understood.The intracellular signaling pathways activated by PTH and PTHrP via the PTH1R receptor include G s -mediated activation of adenylate cyclase, resulting in cAMP production and PKA activation, and G q/11 -mediated PLC␤ stimulation, leading to inositol 1,4,5-trisphosphate (IP 3 ) production, calcium mobilization, and PKC activation (4 -7). It has also been demonstrated that PTH activates the Raf-MEK-ERK MAP kinase (MAPK) cascade through both PKA and PKC in a cell-specific and G protein-dependent manner (8 -10). MAPKs activated in response to stimulation by many different classes of cell surface receptors, including growth factor receptor tyrosine kinases and 7TMRs, regulate cell growth, division, differentiation, and apoptosis (11). PTHstimulated activation of MAPK is known to have proliferative effects in kidney and bone (12, 13).There is growing evidence for novel 7TMR signaling mechanisms that are distinct from the classical G protein second messenger-dependent pathways. One such mechanism involves ␤-arrestins, a small family of cytosolic proteins initially identified for their central role in 7TMR desensitization. ␤-Arrestins are recruited to agonist-occupied 7TMRs that have been phosphorylated by specialized G protein-coupled receptor kinases (GRKs) and sterically inhibit receptor-G protein coupling resulting in homologous receptor desensitization. Additionally, ␤-arrestins act as adaptors in clathrin-mediated receptor endocytosis (14, 15). The role of ␤-arrestins acting as signal transducers through the formation of scaffolding complexes with accessory effector molecules such as Src, Ras, ERK1/2, JNK3, and MAPK kinase 4 (MKK4) is becoming increasingly recognized (16 -20).The potential signaling diversity of 7TMRs suggests the possible existence of multiple discrete "active" receptor conformations. This implies that specific ligands might direct distinct signaling responses by preferentially stabilizing one or more of these active conformations. In the simple two-state model of receptor activation, agonists are defined as drugs that stabilize the active receptor conformation, which in turn promotes G protein activation. Conversely, an inverse agonist preferentially binds to the inactive receptor conformational state thereby reduc-