title: Differences in endosomal targeting of human (beta)1- and (beta)2-adrenergic receptors following clathrin-mediated endocytosis.
abstract: The beta(2)-adrenergic receptor (beta(2)AR) undergoes agonist-mediated endocytosis via clathrin-coated pits by a process dependent on both arrestins and dynamin. Internalization of some G protein-coupled receptors, however, is independent of arrestins and/or dynamin and through other membrane microdomains such as caveolae or lipid rafts. The human beta(1)AR is less susceptible to agonist-mediated internalization than the beta(2)-subtype, and its endocytic route, which is unknown, may be different. We have found that (i) co-expression of arrestin-2 or -3 enhanced the internalization of both subtypes whereas co-expression of dominant-negative mutants of arrestin-2 or dynamin impaired their internalization, as did inhibitors of clathrin-mediated endocytosis. (ii) Agonist stimulation increased the phosphorylation of beta(2)AR but not beta(1)AR. (iii) In response to agonist, each subtype redistributed from the cell surface to a distinct population of cytoplasmic vesicles; those containing beta(1)AR were smaller and closer to the plasma membrane whereas those containing beta(2)AR were larger and more perinuclear. (iv) When subcellular fractions from agonist-treated cells were separated by sucrose density gradient centrifugation, all of the internalized beta(2)AR appeared in the lighter endosomal-containing fractions whereas some of the internalized beta(1)AR remained in the denser plasma membrane-containing fractions. (v) Both subtypes recycled with similar kinetics back to the cell surface upon removal of agonist; however, recycling of beta(2)AR but not beta(1)AR was inhibited by monensin. Based on these results, we propose that the internalization of beta(1)AR is both arrestin- and dynamin-dependent and follows the same clathrin-mediated endocytic pathway as beta(2)AR. But during or after endocytosis, beta(1)AR and beta(2)AR are sorted into different endosomal compartments.
authors.affiliation: Membrane Biochemistry Section, Laboratory of Molecular and Cellular Neurobiology, National Institute of Neurological Disorders and Stroke, The National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Liang
authors.forename: Patricia K
authors.initials: PK
authors.lastname: Curran
authors.forename: Quang
authors.initials: Q
authors.lastname: Hoang
authors.forename: R Travis
authors.initials: RT
authors.lastname: Moreland
authors.forename: Peter H
authors.initials: PH
authors.lastname: Fishman
mesh_terms: D058665:Adrenergic beta-1 Receptor Agonists; D058666:Adrenergic beta-2 Receptor Agonists; D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology; D002966:Clathrin / Q000502:physiology*; D006224:Cricetinae; D034281:Dynamins / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000378:metabolism*; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D018942:Macrolides / Q000494:pharmacology; D008985:Monensin / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D021381:Protein Transport / Q000187:drug effects; D018342:Receptors, Adrenergic, beta-1 / Q000235:genetics / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology
chemical_list: D058665:Adrenergic beta-1 Receptor Agonists; D058666:Adrenergic beta-2 Receptor Agonists; D019390:Arrestins; D002966:Clathrin; D006982:Hypertonic Solutions; D018942:Macrolides; D018342:Receptors, Adrenergic, beta-1; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D013395:Sucrose; C036978:concanamycin A; D002772:Cholera Toxin; D008985:Monensin; D034281:Dynamins; D007545:Isoproterenol