title: Differences in the signaling pathways of α(1A)- and α(1B)-adrenoceptors are related to different endosomal targeting.
abstract: OBJECTIVE
  To compare the constitutive and agonist-dependent endosomal trafficking of α(1A)- and α(1B)-adrenoceptors (ARs) and to establish if the internalization pattern determines the signaling pathways of each subtype.
  
  
  METHODS
  Using CypHer5 technology and VSV-G epitope tagged α(1A)- and α(1B)-ARs stably and transiently expressed in HEK 293 cells, we analyzed by confocal microscopy the constitutive and agonist-induced internalization of each subtype, and the temporal relationship between agonist induced internalization and the increase in intracellular calcium (determined by FLUO-3 flouorescence), or the phosphorylation of ERK1/2 and p38 MAP kinases (determined by Western blot).
  
  
  CONCLUSIONS
  Constitutive as well as agonist-induced trafficking of α(1A) and α(1B) ARs maintain two different endosomal pools of receptors: one located close to the plasma membrane and the other deeper into the cytosol. Each subtype exhibited specific characteristics of internalization and distribution between these pools that determines their signaling pathways: α(1A)-ARs, when located in the plasma membrane, signal through calcium and ERK1/2 pathways but, when translocated to deeper endosomes, through a mechanism sensitive to β-arrestin and concanavalin A, continue signaling through ERK1/2 and also activate the p38 pathway. α(1B)-ARs signal through calcium and ERK1/2 only when located in the membrane and the signals disappear after endocytosis and by disruption of the membrane lipid rafts by methyl-β-cyclodextrin.
authors.affiliation: Departamento de Farmacología, Facultad de Farmacia, Universitat de València, Valencia, Spain.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Segura
authors.forename: Miguel
authors.initials: M
authors.lastname: Pérez-Aso
authors.forename: Fermí
authors.initials: F
authors.lastname: Montó
authors.forename: Elena
authors.initials: E
authors.lastname: Carceller
authors.forename: María Antonia
authors.initials: MA
authors.lastname: Noguera
authors.forename: John
authors.initials: J
authors.lastname: Pediani
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
authors.forename: Ian Christie
authors.initials: IC
authors.lastname: McGrath
authors.forename: Pilar
authors.initials: P
authors.lastname: D'Ocon
mesh_terms: D058646:Adrenergic alpha-1 Receptor Agonists / Q000494:pharmacology; D020013:Calcium Signaling; D002460:Cell Line; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D058646:Adrenergic alpha-1 Receptor Agonists; D018340:Receptors, Adrenergic, alpha-1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases