Cellular Trafficking of G Protein-coupled Receptor/β-Arrestin Endocytic Complexes

Abstract

␤-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 ...

A cluster of differentially expressed signal transduction genes identified by microarray analysis in a rat genetic model of alcoholism

“…16 This mechanism has been demonstrated for the alpha1b-and beta2-adrenergic receptors, muand delta-opioid receptors, the dopamine D1 receptor and cannabinoid CB1 receptor. 17,18 Upon ligand binding b-arrestin also recruits cAMP phosphodiesterase type IV to the recetor-adenylate cyclase complex. 19 Both mechanisms lead to receptor desensitization and cAP signal termination.…”

Intracellular angiotensin II activates rat myometrium

“…Intracellular GPCRs are either located at the Golgi, where they undergo modifications that will further allow insertion and functionality at the plasma membrane, or targeted at the endolysosomal compartment upon internalization. The AT 1A receptor is known to remain associated with ␤-arrestin upon endocytosis (2,47), being further trafficked to cytosolic vesicular structures; however, it is neither dephosphorylated nor efficiently recycled back to the cell surface (2). In our experiments, angiotensin II-induced Ca 2ϩ response was not affected by brefeldin A, a Golgi apparatus disruptor (4); however, pretreatment with bafilomycin A 1 , a V-type ATPase inhibitor that prevents endo-lysosomal acidification (6), abolished the response.…”

“…Extracellular angiotensin II induces dose-dependent contractions of the myometrium (12,32,44). This effect is sensitive to the AT 1 blocker losartan and depends on the estrous cycle (1,47). Since an increase in [Ca 2ϩ ] i is essential in uterine contractility, in this study we assessed the effects of intracellular injection of angiotensin II on [Ca 2ϩ ] i using calcium imaging.…”

Constitutive arrestin-mediated desensitization of a human vasopressin receptor mutant associated with nephrogenic diabetes insipidus

“…The increased affinity of activated receptors for arrestins results in the observable translocation of arrestins from the cell cytosol to the plasma membrane, frequently followed by clathrin-coated pit internalization of the arrestin-bound receptors (5)(6)(7)(8)(9). Arrestin translocation has been visualized by using fusion proteins of green fluorescent protein (GFP) homologues and an arrestin family member (visual arrestin, ␤arrestin1 and ␤arres-tin2) for numerous GPCRs including the angiotensin, neurokinin, thyrotropin-releasing hormone, and human vasopressin type II receptors (V2R) (5,6,(10)(11)(12).…”