␤-Arrestins are multifunctional proteins identified on the basis of their ability to bind and uncouple G proteincoupled receptors (GPCR) from heterotrimeric G proteins. In addition, ␤-arrestins play a central role in mediating GPCR endocytosis, a key regulatory step in receptor resensitization. In this study, we visualize the intracellular trafficking of ␤-arrestin2 in response to activation of several distinct GPCRs including the ␤ 2 -adrenergic receptor (␤ 2 AR), angiotensin II type 1A receptor (AT 1A R), dopamine D1A receptor (D 1A R), endothelin type A receptor (ET A R), and neurotensin receptor (NTR). Our results reveal that in response to ␤ 2 AR activation, ␤-arrestin2 translocation to the plasma membrane shares the same pharmacological profile as described for receptor activation and sequestration, consistent with a role for ␤-arrestin as the agonistdriven switch initiating receptor endocytosis. Whereas redistributed ␤-arrestins are confined to the periphery of cells and do not traffic along with activated ␤ 2 AR, D 1A R, and ET A R in endocytic vesicles, activation of AT 1A R and NTR triggers a clear time-dependent redistribution of ␤-arrestins to intracellular vesicular compartments where they colocalize with internalized receptors. Activation of a chimeric AT 1A R with the ␤ 2 AR carboxyl-terminal tail results in a ␤-arrestin membrane localization pattern similar to that observed in response to ␤ 2 AR activation. In contrast, the corresponding chimeric ␤ 2 AR with the AT 1A R carboxyl-terminal tail gains the ability to translocate ␤-arrestin to intracellular vesicles. These results demonstrate that the cellular trafficking of ␤-arrestin proteins is differentially regulated by the activation of distinct GPCRs. Furthermore, they suggest that the carboxyl-tail of the receptors might be involved in determining the stability of receptor/␤-arrestin complexes and cellular distribution of ␤-arrestins.Signal transduction via G protein-coupled receptors (GPCRs) 1 is intimately associated with a wide variety of biological processes including neurotransmission, chemoattraction, cardiac function, olfaction, and vision. ␤-Arrestin proteins play an important role in regulating the responsiveness of GPCRs by contributing to mechanisms involved in both GPCR desensitization and resensitization (1-5). ␤-Arrestins regulate GPCR desensitization by binding and uncoupling the receptors from heterotrimeric G proteins once they have been phosphorylated by G protein-coupled receptor kinases (GRKs) (1, 3). In addition, they are also required for the sequestration (endocytosis) of a growing number of GPCRs, including the CCR-5, follitropin receptor, lutropin/choriogonadotropin receptor, m2 muscarinic acetylcholine receptor, mu opioid receptor, substance P receptor, and the ␤ 2 -adrenergic receptor (␤ 2 AR) (4, 6 -11). At least in the case of the ␤ 2 AR, the agonist-dependent sequestration of the receptor to an endosomal compartment not only promotes receptor dephosphorylation but is essential for the re-establishment of normal ...

beta-Arrestins are important in chemoattractant receptor-induced granule release, a process that may involve Ral-dependent regulation of the actin cytoskeleton. We have identified the Ral GDP dissociation stimulator (Ral-GDS) as a beta-arrestin-binding protein by yeast two-hybrid screening and co-immunoprecipitation from human polymorphonuclear neutrophilic leukocytes (PMNs). Under basal conditions, Ral-GDS is localized to the cytosol and remains inactive in a complex formed with beta-arrestins. In response to formyl-Met-Leu-Phe (fMLP) receptor stimulation, beta-arrestin Ral-GDS protein complexes dissociate and Ral-GDS translocates with beta-arrestin from the cytosol to the plasma membrane, resulting in the Ras-independent activation of the Ral effector pathway required for cytoskeletal rearrangement. The subsequent re-association of beta-arrestin Ral-GDS complexes is associated with the inactivation of Ral signalling. Thus, beta-arrestins regulate multiple steps in the Ral-dependent processes that result in chemoattractant-induced cytoskeletal reorganization.

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Metabotropic glutamate receptors (mGluRs) constitute a unique subclass of G protein-coupled receptors (GPCRs) that bear little sequence homology to other members of the GPCR superfamily. The mGluR subtypes that are coupled to the hydrolysis of phosphoinositide contribute to both synaptic plasticity and glutamatemediated excitotoxicity in neurons. In the present study, the expression of mGluR1a in HEK 293 cells led to agonist-independent cell death. Since G protein-coupled receptor kinases (GRKs) desensitize a diverse variety of GPCRs, we explored whether GRKs contributed to the regulation of both constitutive and agonist-stimulated mGluR1a activity and thereby may prevent mGluR1a-mediated excitotoxicity associated with mGluR1a overactivation. We find that the co-expression of mGluR1a with GRK2 and GRK5, but not GRK4 and GRK6, reduced both constitutive and agonist-stimulated mGluR1a activity. Agonist-stimulated mGluR1a phosphorylation was enhanced by the co-expression of GRK2 and was blocked by two different GRK2 dominant-negative mutants. Furthermore, GRK2-dependent mGluR1a desensitization protected against mGluR1a-mediated cell death, at least in part by blocking mGluR1a-stimulated apoptosis. Our data indicate that as with other members of the GPCR superfamily, a member of the structurally distinct mGluR family (mGluR1a) serves as a substrate for GRK-mediated phosphorylation and that GRK-dependent "feedback" modulation of mGluR1a responsiveness protects against pathophysiological mGluR1a signaling.Glutamate is the major excitatory neurotransmitter in the central nervous system and is essential in the regulation of brain functions and neural cell development (1, 2). Receptors that respond to glutamate are classified into two types, ionotropic and metabotropic. The ionotropic glutamate receptors are cation-specific ion channels that mediate fast excitatory glutamate responses and are subdivided into ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate and NMDA 1 receptors (1, 2). In contrast, metabotropic glutamate receptors (mGluRs) are members of the G protein-coupled receptor (GPCR) superfamily and mediate slower glutamate responses by coupling to various second messenger cascades via heterotrimeric G proteins (1, 2). This property allows mGluRs to translate relatively short neuronal activation into long lasting changes in synaptic activity. As a consequence, mGluR signaling plays an important role in the processes underlying synaptic plasticity (e.g. memory and learning) (3).The mGluR family of receptors constitute a unique subclass of GPCRs that bear no sequence or structural homology to prototypic Class 1 (rhodopsin, ␤ 2 -adrenergic family) and Class 2 (secretin family) GPCRs other than the retention of the seven transmembrane spanning domain topology that is characteristic of a GPCR (4). Furthermore, unlike other GPCR family members, mGluRs couple to heterotrimeric G proteins via the second intracellular loop domain of the receptor rather than the third intracellular loop domain (5). The mGl...

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