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Nuclear localization of Formyl-Peptide Receptor 2 in human cancer cells

Author:
Cattaneo, Fabio, Parisi, Melania, Fioretti, Tiziana, Sarnataro, Daniela, Esposito, Gabriella, Ammendola, Rosario
Source:
Archives of biochemistry and biophysics 2016 v.603 pp. 10-19
ISSN:
0003-9861
Subject:
G-protein coupled receptors, Western blotting, agonists, fluorescent antibody technique, humans, models, mutagenesis, neoplasm cells, neoplasms, nuclear localization signals, phosphorylation
Abstract:
Current models of G protein-coupled receptors (GPCRs) signaling describe binding of external agonists to cell surface receptors which, in turn, trigger several biological responses. New paradigms indicate that GPCRs localize to and signal at the nucleus, thus regulating distinct signaling cascades. The formyl-peptide receptor FPR2 belongs to the GPCR super-family and is coupled to PTX-sensitive Gi proteins. We show by western blot analysis, immunofluorescence experiments and radioligand binding assays that FPR2 is expressed at nuclear level in CaLu-6 and AGS cells. Nuclear FPR2 is a functional receptor, since it participates in intra-nuclear signaling, as assessed by decreased G protein-FPR2 association and enhanced ERK2, c-Jun and c-Myc phosphorylation upon stimulation of intact nuclei with the FPR2 agonist, WKYMVm. We analyzed FPR2 sequence for the search of a nuclear localization sequence (NLS) and we found a stretch of basic aminoacids (227-KIHKK-231) in the third cytoplasmic loop of the receptor. We performed single (K230A) and multiple (H229A/K230A/K231A) mutagenesis of NLS. The constructs were individually overexpressed in HEK293 cells and immunofluorescence and western blot analysis showed that nuclear localization or translocation of FPR2 depends on the integrity of the H229 and K231 residues within the NLS.
Agid:
5247714