title: Agonist-independent nuclear localization of the Apelin, angiotensin AT1, and bradykinin B2 receptors.
abstract: Signaling of the apelin, angiotensin, and bradykinin peptides is mediated by G protein-coupled receptors related through structure and similarities of physiological function. We report nuclear expression as a characteristic of these receptors, including a nuclear localization for the apelin receptor in brain and cerebellum-derived D283 Med cells and the AT(1) and bradykinin B(2) receptors in HEK-293T cells. Immunocytochemical analyses revealed the apelin receptor with localization in neuronal nuclei in cerebellum and hypothalamus, exhibiting expression in neuronal cytoplasm or in both nuclei and cytoplasm. Confocal microscopy of HEK-293T cells revealed the majority of transfected cells displayed constitutive nuclear localization of AT(1) and B(2) receptors, whereas apelin receptors did not show nuclear localization in these cells. The majority of apelin receptor-transfected cerebellum D283 Med cells showed receptor nuclear expression. Immunoblot analyses of subcellular-fractionated D283 Med cells demonstrated endogenous apelin receptor species in nuclear fractions. In addition, an identified nuclear localization signal motif in the third intracellular loop of the apelin receptor was disrupted by a substituted glutamine in place of lysine. This apelin receptor (K242Q) did not exhibit nuclear localization in D283 Med cells. These results demonstrate the following: (i) the apelin receptor exhibits nuclear localization in human brain; (ii) distinct cell-dependent mechanisms for the nuclear transport of apelin, AT(1), and B(2) receptors; and (iii) the disruption of a nuclear localization signal sequence disrupts the nuclear translocation of the apelin receptor. This discovery of apelin, AT(1), and B(2) receptors with agonist-independent nuclear translocation suggests major unanticipated roles for these receptors in cell signaling and function.
authors.affiliation: Department of Pharmacology, University of Toronto, Toronto, Ontario M5S 1A8, USA.
authors.forename: Dennis K
authors.initials: DK
authors.lastname: Lee
authors.forename: A José
authors.initials: AJ
authors.lastname: Lança
authors.forename: Regina
authors.initials: R
authors.lastname: Cheng
authors.forename: Tuan
authors.initials: T
authors.lastname: Nguyen
authors.forename: Xiao Dong
authors.initials: XD
authors.lastname: Ji
authors.forename: Fernand
authors.initials: F
authors.lastname: Gobeil
authors.forename: Sylvain
authors.initials: S
authors.lastname: Chemtob
authors.forename: Susan R
authors.initials: SR
authors.lastname: George
authors.forename: Brian F
authors.initials: BF
authors.lastname: O'Dowd
mesh_terms: D000818:Animals; D000075244:Apelin Receptors; D001921:Brain / Q000648:ultrastructure; D019556:COS Cells; D002458:Cell Fractionation; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry*; D002531:Cerebellum / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000737:chemistry; D004622:Embryo, Mammalian; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D007031:Hypothalamus / Q000648:ultrastructure; D007150:Immunohistochemistry; D007668:Kidney; D008164:Luminescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D009474:Neurons / Q000648:ultrastructure; D021382:Protein Sorting Signals; D051381:Rats; D044140:Receptor, Angiotensin, Type 1 / Q000032:analysis* / Q000235:genetics; D043782:Receptor, Bradykinin B2 / Q000032:analysis* / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000032:analysis* / Q000235:genetics; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D014162:Transfection
chemical_list: C104575:APLNR protein, human; D000075244:Apelin Receptors; C491284:Aplnr protein, rat; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D044140:Receptor, Angiotensin, Type 1; D043782:Receptor, Bradykinin B2; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins