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.
| Original language | English |
|---|---|
| Pages (from-to) | 10-19 |
| Number of pages | 10 |
| Journal | Archives of Biochemistry and Biophysics |
| Volume | 603 |
| DOIs | |
| Publication status | Published - Aug 1 2016 |
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Keywords
- FPR2
- G protein-coupled receptors
- Nuclear localization
- Signal transduction
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
Cite this
Nuclear localization of Formyl-Peptide Receptor 2 in human cancer cells. / Cattaneo, Fabio; Parisi, Melania; Fioretti, Tiziana; Sarnataro, Daniela; Esposito, Gabriella; Ammendola, Rosario.
In: Archives of Biochemistry and Biophysics, Vol. 603, 01.08.2016, p. 10-19.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Nuclear localization of Formyl-Peptide Receptor 2 in human cancer cells
AU - Cattaneo, Fabio
AU - Parisi, Melania
AU - Fioretti, Tiziana
AU - Sarnataro, Daniela
AU - Esposito, Gabriella
AU - Ammendola, Rosario
PY - 2016/8/1
Y1 - 2016/8/1
N2 - 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.
AB - 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.
KW - FPR2
KW - G protein-coupled receptors
KW - Nuclear localization
KW - Signal transduction
UR - http://www.scopus.com/inward/record.url?scp=84968830349&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84968830349&partnerID=8YFLogxK
U2 - 10.1016/j.abb.2016.05.006
DO - 10.1016/j.abb.2016.05.006
M3 - Article
VL - 603
SP - 10
EP - 19
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
SN - 0003-9861
ER -