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      MARCH2 promotes endocytosis and lysosomal sorting of carvedilol-bound β 2-adrenergic receptors

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          Abstract

          The β 2-adrenergic receptor antagonist carvedilol recruits MARCH2, a unique E3 ubiquitin ligase, to promote receptor endocytosis and lysosomal trafficking.

          Abstract

          Lysosomal degradation of ubiquitinated β 2-adrenergic receptors (β 2ARs) serves as a major mechanism of long-term desensitization in response to prolonged agonist stimulation. Surprisingly, the βAR antagonist carvedilol also induced ubiquitination and lysosomal trafficking of both endogenously expressed β 2ARs in vascular smooth muscle cells (VSMCs) and overexpressed Flag-β 2ARs in HEK-293 cells. Carvedilol prevented β 2AR recycling, blocked recruitment of Nedd4 E3 ligase, and promoted the dissociation of the deubiquitinases USP20 and USP33. Using proteomics approaches (liquid chromatography–tandem mass spectrometry), we identified that the E3 ligase MARCH2 interacted with carvedilol-bound β 2AR. The association of MARCH2 with internalized β 2ARs was stabilized by carvedilol and did not involve β-arrestin. Small interfering RNA–mediated down-regulation of MARCH2 ablated carvedilol-induced ubiquitination, endocytosis, and degradation of endogenous β 2ARs in VSMCs. These findings strongly suggest that specific ligands recruit distinct E3 ligase machineries to activated cell surface receptors and direct their intracellular itinerary. In response to β blocker therapy with carvedilol, MARCH2 E3 ligase activity regulates cell surface β 2AR expression and, consequently, its signaling.

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          Most cited references64

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          G protein pathways.

          The heterotrimeric guanine nucleotide-binding proteins (G proteins) are signal transducers that communicate signals from many hormones, neurotransmitters, chemokines, and autocrine and paracrine factors. The extracellular signals are received by members of a large superfamily of receptors with seven membrane-spanning regions that activate the G proteins, which route the signals to several distinct intracellular signaling pathways. These pathways interact with one another to form a network that regulates metabolic enzymes, ion channels, transporters, and other components of the cellular machinery controlling a broad range of cellular processes, including transcription, motility, contractility, and secretion. These cellular processes in turn regulate systemic functions such as embryonic development, gonadal development, learning and memory, and organismal homeostasis.
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            Distinct phosphorylation sites on the β(2)-adrenergic receptor establish a barcode that encodes differential functions of β-arrestin.

            Phosphorylation of G protein-coupled receptors (GPCRs, which are also known as seven-transmembrane spanning receptors) by GPCR kinases (GRKs) plays essential roles in the regulation of receptor function by promoting interactions of the receptors with β-arrestins. These multifunctional adaptor proteins desensitize GPCRs, by reducing receptor coupling to G proteins and facilitating receptor internalization, and mediate GPCR signaling through β-arrestin-specific pathways. Detailed mapping of the phosphorylation sites on GPCRs targeted by individual GRKs and an understanding of how these sites regulate the specific functional consequences of β-arrestin engagement may aid in the discovery of therapeutic agents targeting individual β-arrestin functions. The β(2)-adrenergic receptor (β(2)AR) has many serine and threonine residues in the carboxyl-terminal tail and the intracellular loops, which are potential sites of phosphorylation. We monitored the phosphorylation of the β(2)AR at specific sites upon stimulation with an agonist that promotes signaling by both G protein-mediated and β-arrestin-mediated pathways or with a biased ligand that promotes signaling only through β-arrestin-mediated events in the presence of the full complement of GRKs or when either GRK2 or GRK6 was depleted. We correlated the specific and distinct patterns of receptor phosphorylation by individual GRKs with the functions of β-arrestins and propose that the distinct phosphorylation patterns established by different GRKs establish a "barcode" that imparts distinct conformations to the recruited β-arrestin, thus regulating its functional activities.
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              Arrestin-related ubiquitin-ligase adaptors regulate endocytosis and protein turnover at the cell surface.

              The diversity of plasma membrane (PM) proteins presents a challenge for the achievement of cargo-specific regulation of endocytosis. Here, we describe a family of proteins in yeast (ARTs, for arrestin-related trafficking adaptors) that function by targeting specific PM proteins to the endocytic system. Two members (Art1 and Art2) of the family were discovered in chemical-genetic screens, and they direct downregulation of distinct amino acid transporters triggered by specific stimuli. Sequence analysis revealed a total of nine ART family members in yeast. In addition to similarity to arrestins, the ARTs each contain multiple PY motifs. These motifs are required for recruitment of the Rsp5/Nedd4-like ubiquitin ligase, which modifies the cargoes as well as the ARTs. As a result, ubiquitinated cargoes are internalized and targeted to the vacuole (lysosome) for degradation. We propose that ARTs provide a cargo-specific quality-control pathway that mediates endocytic downregulation by coupling Rsp5/Nedd4 to diverse plasma membrane proteins.
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                Author and article information

                Journal
                J Cell Biol
                J. Cell Biol
                jcb
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                26 November 2012
                : 199
                : 5
                : 817-830
                Affiliations
                [1 ]Department of Medicine and [2 ]Department of Cell Biology, Duke University Medical Center, Durham, NC 27710
                [3 ]Department of Biological Sciences, Tokyo Institute of Technology, Yokohama 226-8501, Japan
                Author notes
                Correspondence to Sudha K. Shenoy: sudha@ 123456receptor-biol.duke.edu
                Article
                201208192
                10.1083/jcb.201208192
                3514787
                23166351
                aae1d0e0-f7da-45db-a791-a0a07f9049bc
                © 2012 Han et al.

                This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

                History
                : 31 August 2012
                : 18 October 2012
                Categories
                Research Articles
                Article

                Cell biology
                Cell biology

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