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Ligand-specific conformational change of the G-protein-coupled receptor ALX/FPR2 determines proresolving functional responses

Cooray, SN; Gobbetti, T; Montero-Melendez, T; McArthur, S; Thompson, D; Clark, AJL; Flower, RJ; Perretti, M (2013) Ligand-specific conformational change of the G-protein-coupled receptor ALX/FPR2 determines proresolving functional responses. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 110 (45). 18232 - 18237 (6). ISSN 0027-8424 https://doi.org/10.1073/pnas.1308253110

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Abstract

Formyl-peptide receptor type 2 (FPR2), also called ALX (the lipoxin A4 receptor), conveys the proresolving properties of lipoxin A4 and annexin A1 (AnxA1) and the proinflammatory signals elicited by serum amyloid protein A and cathelicidins, among others. We tested here the hypothesis that ALX might exist as homo- or heterodimer with FPR1 or FPR3 (the two other family members) and operate in a ligand-biased fashion. Coimmunoprecipitation and bioluminescence resonance energy transfer assays with transfected HEK293 cells revealed constitutive dimerization of the receptors; significantly, AnxA1, but not serum amyloid protein A, could activate ALX homodimers. A p38/MAPK-activated protein kinase/heat shock protein 27 signaling signature was unveiled after AnxA1 application, leading to generation of IL-10, as measured in vitro (in primary monocytes) and in vivo (after i.p. injection in the mouse). The latter response was absent in mice lacking the ALX ortholog. Using a similar approach, ALX/FPR1 heterodimerization evoked using the panagonist peptide Ac2-26, identified a JNK-mediated proapoptotic path that was confirmed in primary neutrophils. These findings provide a molecular mechanism that accounts for the dual nature of ALX and indicate that agonist binding and dimerization state contribute to the conformational landscape of FPRs. G-protein–coupled receptors (GPCRs) constitute a large family of cell surface receptors that share structural characteristics and perform pivotal biological functions, transducing signals from hormones, autacoids, and chemokines. The human GPCR termed “ALX/FPR2” (formyl peptide receptor type 2 or lipoxin A4 receptor, hereafter referred to as “ALX”) is a unique GPCR, shown to convey signals induced by proteins, peptides, and lipid ligands (1). ALX belongs to a small family of receptors that is also activated by formylated peptides, short amino acid sequences with an N-terminal formyl group released by pathogenic and commensal bacteria, as well as by mitochondria upon cell damage. There are three human FPRs and they are termed FPR1, ALX, and FPR3 (2). In view of their different nature and potential engagement with a large number endogenous and exogenous ligands, elucidation of FPR functions may reveal important biological pathways. ALX is an unconventional receptor for the diversity of its agonists and because it can convey contrasting biological signals. The proresolving and anti-inflammatory properties of the protein annexin A1 (AnxA1) and the lipid lipoxin A4 (LXA4), which include neutrophil apoptosis and macrophage efferocytosis, are mediated by this receptor, as shown using pharmacological approaches (1, 3) and more recently with knockout mouse models (4). At the same time, the proinflammatory responses elicited by the cathelicidin-associated antimicrobial peptide LL-37 and serum amyloid protein A (SAA) are also mediated by ALX, which modulates leukocyte activation, recruitment to the site of inflammation, and lifespan (5–7). Moreover, LXA4 and AnxA1 engage ALX to favor a macrophage M2 phenotype, whereas LL-37 and SAA used the same receptor to induce an M1 phenotype (8). In a similar vein using human neutrophils, nanomolar concentrations of LXA4 counteracted LL-37–mediated release of leukotriene B4, both actions being conveyed by ALX (9). Using resonance energy transfer (RET) techniques to investigate GPCR interactions, a number of studies reported agonist stimulation to initiate dimerization between certain GPCRs, which may otherwise exist as monomeric structures (10) or that agonists enhance the interaction between preformed dimers (11). Agonist-induced changes in RET signal could be attributed to conformational changes occurring within the receptor in response to agonist binding (12). Finally, activation of downstream signaling pathways may also influence conformational changes within the receptors (13, 14). We tested here whether the ability of ALX to transduce the bioactions elicited by distinct agonists is modulated by conformational changes.

Item Type: Article
Additional Information: PubMed ID: 24108355
Keywords: inflammation, leukocyte, resolution signaling, Amino Acid Sequence, Animals, Annexin A1, Bioluminescence Resonance Energy Transfer Techniques, Dimerization, HEK293 Cells, Humans, Immunoprecipitation, Interleukin-10, Mice, Molecular Sequence Data, Protein Conformation, Receptors, Formyl Peptide, Serum Amyloid A Protein, Signal Transduction, Science & Technology, Multidisciplinary Sciences, Science & Technology - Other Topics, MULTIDISCIPLINARY SCIENCES, inflammation, leukocyte, resolution signaling, FORMYL-PEPTIDE RECEPTOR, HEAT-SHOCK-PROTEIN, SERUM-AMYLOID-A, LIPOXIN A(4), HUMAN NEUTROPHILS, ANNEXIN A1, TNF-ALPHA, INFLAMMATION, RESOLUTION, APOPTOSIS
Journal or Publication Title: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
ISSN: 0027-8424
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Dates:
DateEvent
5 November 2013Published
Web of Science ID: WOS:000326550800054
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URI: https://openaccess.sgul.ac.uk/id/eprint/104862
Publisher's version: https://doi.org/10.1073/pnas.1308253110

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