Differential mechanism for the cell surface sorting and agonist‐promoted internalization of the α<sub>1B</sub>‐adrenoceptor

Hirasawa, Akira; Awaji, Takeo; Sugawara, Tatsuo; Tsujimoto, Aiko; Tsujimoto, Gozoh

doi:10.1038/sj.bjp.0701795

Cited by 31 publications

(23 citation statements)

References 33 publications

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“…This is in agreement with the results of a previous study showing that actin depolymerization caused missorting of the ␤2-AR to lysosomes instead of recycling vesicles (10). In contrast with our findings, a previous study reported that cytochalasin D could inhibit the internalization of the ␣1b-AR in Chinese hamster ovary cells (26). However, several differences exist between the two studies, including the fact that in Chinese hamster ovary cells the internalization of the ␣1b-AR seemed to be clathrin-independent, whereas we have recently reported that in HEK-293 cells it involves clathrin-coated pits (6).…”

Section: Discussioncontrasting

confidence: 57%

Ezrin Directly Interacts with the α1b-Adrenergic Receptor and Plays a Role in Receptor Recycling

Stanasila¹,

Abuin²,

Diviani³

et al. 2006

Journal of Biological Chemistry

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Using the yeast two-hybrid system, we identified ezrin as a protein interacting with the C-tail of the ␣1b-adrenergic receptor (AR). The interaction was shown to occur in vitro between the receptor C-tail and the N-terminal portion of ezrin, or Four-point-one ERM (FERM) domain. The ␣1b-AR/ezrin interaction occurred inside the cells as shown by the finding that the transfected ␣1b-AR and FERM domain or ezrin could be coimmunoprecipitated from human embryonic kidney 293 cell extracts. Mutational analysis of the ␣1b-AR revealed that the binding site for ezrin involves a stretch of at least four arginines on the receptor C-tail. The results from both receptor biotinylation and immunofluorescence experiments indicated that the FERM domain impaired ␣1b-AR recycling to the plasma membrane without affecting receptor internalization. The dominant negative effect of the FERM domain, which relies on its ability to mask the ezrin binding site for actin, was mimicked by treatment of cells with cytochalasin D, an actin depolymerizing agent. A receptor mutant (⌬R8) lacking its binding site in the C-tail for ezrin displayed delayed receptor recycling. These findings identify ezrin as a new protein directly interacting with a G proteincoupled receptor and demonstrate the direct implication of ezrin in GPCR trafficking via an actin-dependent mechanism.G protein-coupled receptors (GPCRs) 2 can act as scaffolds binding a variety of proteins involved in receptor regulation and signaling. Beyond G protein-coupled receptor kinases, ␤-arrestins, and heterotrimeric G protein subunits, a number of proteins have been found to interact with GPCRs using the yeast 2-hybrid system and other biochemical methods (for review, see Ref. 1-3). The functional implications of these interactions are not fully understood; however, they add an increasing complexity to the signaling mechanisms mediated by GPCRs and their regulation.We have previously reported that the ␣1b-adrenergic receptor (AR) undergoes rapid desensitization and endocytosis upon exposure to the agonist (4) and that the agonist-dependent receptor regulation involves both G protein-coupled receptor kinases and ␤-arrestins (5). The molecular determinants involved in agonist-induced regulation of the ␣1b-AR reside within the C-tail of the receptor, as demonstrated by the fact that truncation of this region almost completely abolished receptor desensitization and internalization (4). The ␣1b-AR can internalize in clathrin-coated vesicles, and this phenomenon depends at least in part on the direct binding to the receptor C-tail of the 2 subunit of the AP2 clathrin-adaptor complex (6).To identify new proteins that interact with the ␣1b-AR which could be involved in regulating receptor function, we have used the yeast two-hybrid system and identified ezrin, a member of the ERM (Ezrin, Radixin, Moesin) family of proteins. ERM proteins were originally characterized as structural components of the cell cortex but were soon shown also to participate in signaling pathways (for review, see Ref. 7)....

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Section: Discussioncontrasting

confidence: 57%

Ezrin Directly Interacts with the α1b-Adrenergic Receptor and Plays a Role in Receptor Recycling

Stanasila¹,

Abuin²,

Diviani³

et al. 2006

Journal of Biological Chemistry

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“…Further studies are required to elucidate whether such regulation is a more generalized mechanism that participates in the trafficking of other chemoattractant receptors. As for the regulation of other members of the GPCR superfamily, the conflicting observations regarding the regulation of muscarinic cholinergic receptor and adrenergic receptor trafficking by cytoskeleton components, combined with our present study, suggest that different receptors that belong to the superfamily of GPCR are divergently regulated in this respect (54,59,60). However, it is interesting to note that in similarity to the role of actin filaments in the regulation of CXCR1 and CXCR2 recycling, the recycling process of ␤ 2 -adrenergic receptors was also dependent on actin filaments (54).…”

Section: Discussionmentioning

confidence: 78%

Actin Filaments Are Involved in the Regulation of Trafficking of Two Closely Related Chemokine Receptors, CXCR1 and CXCR2

Zaslaver

Feniger-Barish

Ben‐Baruch

2001

The Journal of Immunology

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The ligand-induced internalization and recycling of chemokine receptors play a significant role in their regulation. In this study, we analyzed the involvement of actin filaments and of microtubules in the control of ligand-induced internalization and recycling of CXC chemokine receptor (CXCR)1 and CXCR2, two closely related G protein-coupled receptors that mediate ELR-expressing CXC chemokine-induced cellular responses. Nocodazole, a microtubule-disrupting agent, did not affect the IL-8-induced reduction in cell surface expression of CXCR1 and CXCR2, nor did it affect the recycling of these receptors following ligand removal and cell recovery at 37°C. In contrast, cytochalasin D, an actin filament depolymerizing agent, promoted the IL-8-induced reduction in cell surface expression of both CXCR1 and CXCR2. Cytochalasin D significantly inhibited the recycling of both CXCR1 and CXCR2 following IL-8-induced internalization, the inhibition being more pronounced for CXCR2 than for CXCR1. Potent inhibition of recycling was observed also when internalization of CXCR2 was induced by another ELR-expressing CXC chemokine, granulocyte chemotactic protein-2. By the use of carboxyl terminus-truncated CXCR1 and CXCR2 it was observed that the carboxyl terminus domains of CXCR1 and CXCR2 were partially involved in the regulation of the actin-mediated process of receptor recycling. The cytochalasin D-mediated inhibition of CXCR2 recycling had a functional relevance because it impaired the ability of CXCR2-expressing cells to mediate cellular responses. These results suggest that actin filaments, but not microtubules, are involved in the regulation of the intracellular trafficking of CXCR1 and CXCR2, and that actin filaments may be required to enable cellular resensitization following a desensitized refractory period.

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“…For example, both latrunculin A and cytochalasin D disrupt actin cytoskeleton, but marked attenuation in internalization of endothelin or epidermal growth factor receptors is seen only with latrunculin A and not with cytochalasin D (8). In contrast, cytochalasin D blocks agonist-induced internalization of ␣ 1B -adrenergic receptor (9), underscoring the complexity of actin dynamics. Considering these contrasting results with chemical agents, we sought to investigate the role of cortical actin cytoskeleton in agonist-mediated ␤ 2 AR internalization by using a chemical agent that disrupts the actin cytoskeleton and mutants of the actin-binding protein cofilin known to alter the integrity of the actin cytoskeleton.…”

mentioning

confidence: 98%

Agonist-stimulated β-Adrenergic Receptor Internalization Requires Dynamic Cytoskeletal Actin Turnover

Volovyk

Wolf

Prasad

et al. 2006

Journal of Biological Chemistry

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Stimulation of ␤-adrenergic receptors (␤ARs) leads to sequential recruitment of ␤-arrestin, AP-2 adaptor protein, clathrin, and dynamin to the receptor complex, resulting in endocytosis. Whether a dynamic actin cytoskeleton is required for ␤AR endocytosis is not known. In this study, we have used ␤ 1 -and ␤ 2 ARs, two ubiquitously expressed members of the ␤AR family, to comprehensively evaluate the requirement of the actin cytoskeleton in receptor internalization. The integrity of the actin cytoskeleton was manipulated with the agent latrunculin B (LB) and mutants of cofilin to depolymerize actin filaments. Treatment of cells with LB resulted in dose-dependent depolymerization of the cortical actin cytoskeleton that was associated with significant attenuation in internalization of ␤ 2 ARs, ␤ 1 ARs, and mutants of ␤ 1 ARs that internalize via either clathrin-or caveolin-dependent pathways. Importantly, LB treatment did not inhibit ␤-arrestin translocation or dynamin recruitment to the agonist-stimulated receptor. To unequivocally demonstrate the requirement of the actin cytoskeleton for ␤ 2 AR endocytosis, we used an actin-binding protein cofilin that biochemically depolymerizes and severs actin filaments. Isoproterenol-mediated internalization of ␤ 2 AR was completely blocked in the presence of wild type cofilin, which could be rescued by a mutant of cofilin that mimics a constitutive phosphorylated state and leads to normal agonist-stimulated ␤ 2 AR endocytosis. Finally, treatment with jasplakinolide, an inhibitor of actin turnover, resulted in dose-dependent inhibition of ␤ 2 AR internalization, suggesting that turnover of actin filaments at the receptor complex is required for endocytosis. Taken together, these data demonstrate that intact and functional dynamic actin cytoskeleton is required for normal ␤AR internalization. ␤-adrenergic receptors (␤ARs)2 belong to the large family of seventransmembrane receptors that are important regulators of cardiac function. In the heart, ␤ARs bind catecholamines and transduce intracellular signals of appropriate magnitude and specificity through tightly regulated mechanisms of receptor activation, desensitization, and internalization (1). ␤AR signaling and the actin cytoskeleton are important for normal cardiac function, and alterations in either system have been implicated in the development of cardiac hypertrophy and failure (2). Agonist-mediated ␤AR internalization is a complex process that is mediated by many proteins, including ␤-arrestin, clathrin, and dynamin (1); however, whether the actin cytoskeleton is essential for ␤ 2 AR internalization is controversial (3).Actin is a critical component of the cytoskeletal network, undergoes dynamic regulation in response to extracellular stimuli, and has been implicated in vesicular trafficking, including endocytosis (4). In budding yeast, actin assembly and disassembly are tightly coupled to vesicle budding and fusion events (5). Furthermore, actin rearrangements in yeast are thought to be essential for receptor endocytosi...

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Differential mechanism for the cell surface sorting and agonist‐promoted internalization of the α_1B‐adrenoceptor

Cited by 31 publications

References 33 publications

Ezrin Directly Interacts with the α1b-Adrenergic Receptor and Plays a Role in Receptor Recycling

Ezrin Directly Interacts with the α1b-Adrenergic Receptor and Plays a Role in Receptor Recycling

Actin Filaments Are Involved in the Regulation of Trafficking of Two Closely Related Chemokine Receptors, CXCR1 and CXCR2

Agonist-stimulated β-Adrenergic Receptor Internalization Requires Dynamic Cytoskeletal Actin Turnover

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Differential mechanism for the cell surface sorting and agonist‐promoted internalization of the α1B‐adrenoceptor

Cited by 31 publications

References 33 publications

Ezrin Directly Interacts with the α1b-Adrenergic Receptor and Plays a Role in Receptor Recycling

Ezrin Directly Interacts with the α1b-Adrenergic Receptor and Plays a Role in Receptor Recycling

Actin Filaments Are Involved in the Regulation of Trafficking of Two Closely Related Chemokine Receptors, CXCR1 and CXCR2

Agonist-stimulated β-Adrenergic Receptor Internalization Requires Dynamic Cytoskeletal Actin Turnover

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Differential mechanism for the cell surface sorting and agonist‐promoted internalization of the α_1B‐adrenoceptor