Parisi, Melania (2016) New insights in Formyl-peptide Receptors functions. [Tesi di dottorato]

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Tipologia del documento: Tesi di dottorato
Lingua: English
Titolo: New insights in Formyl-peptide Receptors functions
Autori:
AutoreEmail
Parisi, Melaniamelania.parisi@unina.it
Data: 30 Marzo 2016
Numero di pagine: 68
Istituzione: Università degli Studi di Napoli Federico II
Dipartimento: Medicina Molecolare e Biotecnologie Mediche
Scuola di dottorato: Medicina molecolare
Dottorato: Biochimica e biologia cellulare e molecolare
Ciclo di dottorato: 28
Coordinatore del Corso di dottorato:
nomeemail
Arcari, Paoloarcari@unina.it
Tutor:
nomeemail
Ammendola, Rosario[non definito]
Data: 30 Marzo 2016
Numero di pagine: 68
Parole chiave: Transactivation; Cell signaling; Nuclear GPCRs
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare
Depositato il: 13 Apr 2016 13:44
Ultima modifica: 10 Mag 2017 01:00
URI: http://www.fedoa.unina.it/id/eprint/10851

Abstract

GPCRs (G-protein-coupled receptors) are versatile signaling molecules at the cell surface and are the largest and most diverse family of membrane receptors in the human genome. They convert a large variety of extracellular stimuliinto intracellular response through the activation of heterotrimeric G-proteins, which constitute the key regulatory elements in a broad range of normal and pathological processes and the most important targets for pharmaceutical drug discovery. The human formyl-peptide receptor FPR1 and its variants FPR2 and FPR3 belong to the Gi-protein coupled seven transmembrane receptor family sensitive to pertussis toxin (PTX)as indicated by the total loss of cell response to their agonists upon exposure to pertussis toxin.They are expressed in several cell types and their crucial biological functions are supported by the identification of high affinity host-derived agonists. An important feature of GPCR is their ability to transactive tyrosine kinase receptor. This cross-communication between different signaling systems plays a key role to coordinate the plethora of extracellular stimuli to which a cell is subjected under several physiological or pathological conditions. We investigated the ability of FPR2 to transactivate the RTK Hepatocyte growth factor receptor (c-Met) in PNT1A cells and of FPR1 to cross-talk with the vascular endothelial growth factor receptor (VEGFR) in ECV cells. We show that WKYMVm an FPR2 agonist induces the phosphorylation of Y1313/1349/1356 of c-Met and the FPR1 agonist N-fMLP triggers VEGFR2 activation by phosphorilation of Y996 residue.As a result of transactivation, phosphotyrosine residues of two receptors provide docking sites for recruitment and triggering of STAT3, PLC-γ/PKCα and PI3K/Akt pathways in both cell lines. The critical role of NADPH oxidase-dependent superoxide generation in this cross-talk mechanism is supported by the finding that blockade of NADPH oxidase function prevents trans-phosphorylation of two TRKs and the resulting downstream signalling cascade. Current model indicate that each GPCR and associated sets of heterotrimeric G proteins on the cell surface had been proven to initiate one or more chains of intracellular signaling events mediated by complexes of GTPases, kinases, and linking proteins.Recentstudies of intracellular trafficking of a few different GPCRs, evoked by exposure to ligand or by cellular activation through ligand-independent mechanisms, have shown that GPCRs may be inserted into nuclear membranes for prolonged periods, compose distinctive signaling units there, and respond to specific intracellular ligand by transducing nuclear transcriptional signals that differ from those sent by their plasma membrane complexes.We show by western blot analysis, immunofluorescence experiments and radioligand binding assays that FPR2, but not FPR1 and FPR3, 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. Taken together, our data indicate that FPRs play new unexepected roles in cell signaling and function. The promiscuity of these receptors in binding different ligands, coupled with their presence in different cells and tissues, indicates a diverse role in multiple biological settings. A better understanding of these fundamental functions could lead to the identification of new therapeutic targets for drug development.

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