title: Identification of a putative nuclear localization sequence within ANG II AT(1A) receptor associated with nuclear activation.
abstract: Angiotensin II (ANG II) type 1 (AT(1)) receptors, similar to other G protein-coupled receptors, undergo desensitization and internalization, and potentially nuclear localization, subsequent to agonist interaction. Evidence suggests that the carboxy-terminal tail may be involved in receptor nuclear localization. In the present study, we examined the carboxy-terminal tail of the receptor for specific regions responsible for the nuclear translocation phenomenon and resultant nuclear activation. Human embryonic kidney cells stably expressing either a wild-type AT(1A) receptor-green fluorescent protein (AT(1A)R/GFP) construct or a site-directed mutation of a putative nuclear localization sequence (NLS) [K307Q]AT(1A)R/GFP (KQ/AT(1A)R/GFP), were examined for differences in receptor nuclear trafficking and nuclear activation. Receptor expression, intracellular signaling, and ANG II-induced internalization of the wild-type/GFP construct and of the KQ/AT(1A)R/GFP mutant was similar. Laser scanning confocal microscopy showed that in cells expressing the AT(1A)R/GFP, trafficking of the receptor to the nuclear area and colocalization with lamin B occurred within 30 min of ANG II (100 nM) stimulation, whereas the KQ/AT(1A)R/GFP mutant failed to demonstrate nuclear localization. Immunoblotting of nuclear lysates with an anti-GFP antibody confirmed these observations. Nuclear localization of the wild-type receptor correlated with increase transcription for both EGR-1 and PTGS-2 genes while the nuclear-deficient KQ/AT(1A)R/GFP mutant demonstrated increases for only the EGR-1 gene. These results suggest that a NLS (KKFKKY; aa307-312) is located within the cytoplasmic tail of the AT(1A) receptor and that nuclear localization of the receptor corresponds with specific activation of transcription for the COX-2 gene PTGS-2.
authors.affiliation: Division of Nephrology, Department of Medicine, Medical University of South Carolina, 829 Clinical Sciences Bldg., 96 Jonathan Lucas St., Charleston, SC 29425, USA. morinelt@musc.edu
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Morinelli
authors.forename: John R
authors.initials: JR
authors.lastname: Raymond
authors.forename: Aleksander
authors.initials: A
authors.lastname: Baldys
authors.forename: Qing
authors.initials: Q
authors.lastname: Yang
authors.forename: Mi-Hye
authors.initials: MH
authors.lastname: Lee
authors.forename: Louis
authors.initials: L
authors.lastname: Luttrell
authors.forename: Michael E
authors.initials: ME
authors.lastname: Ullian
mesh_terms: D021581:Active Transport, Cell Nucleus; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000378:metabolism; D051766:Early Growth Response Protein 1 / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation; D019913:Nuclear Localization Signals / Q000502:physiology*; D017434:Protein Structure, Tertiary; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D008565:Membrane Proteins; D019913:Nuclear Localization Signals; D044140:Receptor, Angiotensin, Type 1; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human