Signaling through ␤-arrestins is a recently appreciated mechanism used by seven-transmembrane receptors. Because G protein-coupled receptor kinase (GRK) phosphorylation of such receptors is generally a prerequisite for ␤-arrestin binding, we studied the roles of different GRKs in promoting ␤-arrestin-mediated extracellular signal-regulated kinase (ERK) activation by a typical seven-transmembrane receptor, the Gs-coupled V2 vasopressin receptor. Gsand ␤-arrestin-mediated pathways to ERK activation could be distinguished with H89, an inhibitor of protein kinase A, and ␤-arrestin 2 small interfering RNA, respectively. The roles of GRK2, -3, -5, and -6 were assessed by suppressing their expression with specific small interfering RNA sequences. By using this approach, we demonstrated that GRK2 and -3 are responsible for most of the agonist-dependent receptor phosphorylation, desensitization, and recruitment of ␤-arrestins. In contrast, GRK5 and -6 mediated much less receptor phosphorylation and ␤-arrestin recruitment, but yet appeared exclusively to support ␤-arrestin 2-mediated ERK activation. GRK2 suppression actually increased ␤-arrestin-stimulated ERK activation. These results suggest that ␤-arrestin recruited in response to receptor phosphorylation by different GRKs has distinct functional potentials. extracellular signal-regulated kinase ͉ phosphorylation ͉ desensitization ͉ small interfering RNA

Binding of Sonic Hedgehog (Shh) to Patched (Ptc) relieves the latter's tonic inhibition of Smoothened (Smo), a receptor that spans the cell membrane seven times. This initiates signaling which, by unknown mechanisms, regulates vertebrate developmental processes. We find that two molecules interact with mammalian Smo in an activation-dependent manner: G protein-coupled receptor kinase 2 (GRK2) leads to phosphorylation of Smo, and beta-arrestin 2 fused to green fluorescent protein interacts with Smo. These two processes promote endocytosis of Smo in clathrin-coated pits. Ptc inhibits association of beta-arrestin 2 with Smo, and this inhibition is relieved in cells treated with Shh. A Smo agonist stimulated and a Smo antagonist (cyclopamine) inhibited both phosphorylation of Smo by GRK2 and interaction of beta-arrestin 2 with Smo. beta-Arrestin 2 and GRK2 are thus potential mediators of signaling by activated Smo.

Wnt proteins bind to seven-transmembrane Frizzled receptors to mediate the important developmental, morphogenetic, and tissue-regenerative effects of Wnt signaling. Dysregulated Wnt signaling is associated with many cancers. Currently there exist no drug candidates, or even tool compounds that modulate Wnt-mediated receptor trafficking, and subsequent Wnt signaling. We examined libraries of FDA-approved drugs for their utility as Frizzled internalization modulators, employing a primary imaged-based GFP-fluorescence assay that uses Frizzled1 endocytosis as the readout. We now report that the anti-helminthic niclosamide, a drug used for the treatment of tapeworm, promotes Frizzled1 endocytosis, down regulates Dishevelled-2 protein, and inhibits Wnt3A-stimulated β-catenin stabilization and LEF/TCF reporter activity. Additionally, following niclosamide mediated internalization, the Frizzled1 receptor co-localizes in vesicles containing Transferrin and agonist-activated β2-adrenergic receptor. Therefore, niclosamide may serve as a negative modulator of Wnt/Frizzled1 signaling by depleting up-stream signaling molecules (i.e. Frizzled and Dishevelled), and moreover may provide a valuable means to study the physiological consequences of Wnt signaling.

␤-Arrestins were initially shown, in conjunction with G protein-coupled receptor kinases, to be involved in the desensitization and internalization of activated seven-transmembrane receptors. Recently, ␤-arrestin 2 has been shown to act as a signal mediator in mitogen-activated protein kinase cascades and to play a positive regulatory role in chemotaxis. We now show that ␤-arrestin 1 is required to activate the small GTPase RhoA leading to the re-organization of stress fibers following the activation of the angiotensin II type 1A receptor. This angiotensin II type 1A receptor-directed RhoA activation and stress fiber formation also require the activation of the heterotrimeric G protein G ␣q/11 . Whereas neither ␤-arrestin 1 nor G ␣q/11 activation alone is sufficient to robustly activate RhoA, the concurrent recruitment of ␤-arrestin 1 and activation of G ␣q/11 leads to full activation of RhoA and to the subsequent formation of stress fibers.

No abstract

The small family of G-protein-coupled receptor kinases (GRKs) regulate cell signaling by phosphorylating heptahelical receptors, thereby promoting receptor interaction with ␤-arrestins. This switches a receptor from G-protein activation to G-protein desensitization, receptor internalization, and ␤-arrestin-dependent signal activation. However, the specificity of GRKs for recruiting ␤-arrestins to specific receptors has not been elucidated. Here we use the ␤ 2 -adrenergic receptor (␤ 2 AR), the archetypal nonvisual heptahelical receptor, as a model to test functional GRK specificity. We monitor endogenous GRK activity with a fluorescence resonance energy transfer assay in live cells by measuring kinetics of the interaction between the ␤ 2 AR and ␤-arrestins. We show that ␤ 2 AR phosphorylation is required for high affinity ␤-arrestin binding, and we use small interfering RNA silencing to show that HEK-293 and U2-OS cells use different subsets of their expressed GRKs to promote ␤-arrestin recruitment, with significant GRK redundancy evident in both cell types. Surprisingly, the GRK specificity for ␤-arrestin recruitment does not correlate with that for bulk receptor phosphorylation, indicating that ␤-arrestin recruitment is specific for a subset of receptor phosphorylations on specific sites. Moreover, multiple members of the GRK family are able to phosphorylate the ␤ 2 AR and induce ␤-arrestin recruitment, with their relative contributions largely determined by their relative expression levels. Because GRK isoforms vary in their regulation, this partially redundant system ensures ␤-arrestin recruitment while providing the opportunity for tissue-specific regulation of the rate of ␤-arrestin recruitment.Arrestins and G-protein-coupled receptor kinases (GRKs) 2 are important regulators of heptahelical receptor function. The two nonvisual arrestins, ␤-arrestin1 and ␤-arrestin2, and the ubiquitous nonvisual GRKs, GRK2, GRK3, GRK5, and GRK6, together form an axis of receptor regulation critical to mammalian physiology (1-3).The ␤-arrestin/GRK system was first described as a means of desensitizing receptors (4), but it is now known to mediate receptor internalization (5) and receptor-stimulated signals as well, including activation of Src, ERK1/2, Rho, and others (6). The primary switch in these regulatory pathways appears to be the recruitment of ␤-arrestins to receptors, a process facilitated by GRK-mediated receptor phosphorylation, which enhances binding affinity for ␤-arrestins (7). ␤-Arrestin binding to phosphorylated receptor sterically hinders G-protein coupling of the receptor (8) and also induces conformational changes in ␤-arrestin (9). These conformational changes regulate the ability of ␤-arrestin to couple to the endocytic machinery (10) and to stimulate ␤-arrestin-dependent signals. Indeed, it appears that ␤-arrestin may adopt functionally distinct conformations depending on specific features of receptor phosphorylation, as siRNA silencing of distinct GRKs has dramatically different effects on receptor in...

␤-Arrestins, originally discovered as terminators of G protein-coupled receptor signaling, have more recently been appreciated to also function as signal transducers in their own right, although the consequences for cellular physiology have not been well understood. Here we demonstrate that ␤-arrestin-2 mediates anti-apoptotic cytoprotective signaling stimulated by a typical 7-transmembrane receptor the angiotensin ATII 1A receptor, expressed endogenously in rat vascular smooth muscle cells or by transfection in HEK-293 cells. Receptor stimulation leads to concerted activation of two pathways, ERK/p90RSK and PI3K/AKT, which converge to phosphorylate and inactivate the pro-apoptotic protein BAD. Anti-apoptotic effects as well as pathway activities can be stimulated by an angiotensin analog (SII), which has been previously shown to activate ␤-arrestin but not G protein-dependent signaling, and are abrogated by ␤-arrestin-2 small interfering RNA. These findings establish a key role for ␤-arrestin-2 in mediating cellular cytoprotective functions by a 7-transmembrane receptor and define the biochemical pathways involved.

Regenerative medicine holds the promise of replacing damaged tissues largely by stem cell activation. Hedgehog signaling through the plasma membrane receptor Smoothened (Smo) is an important process for regulating stem cell proliferation.