Abstract
Formyl peptide receptor 3 (Fpr3, also known as Fpr-rs1) is a G protein-coupled receptor expressed in subsets of sensory neurons of the mouse vomeronasal organ, an olfactory substructure essential for social recognition. Fpr3 has been implicated in the sensing of infection-associated olfactory cues, but its expression pattern and function are incompletely understood. To facilitate visualization of Fpr3-expressing cells, we generated and validated two new anti-Fpr3 antibodies enabling us to analyze acute Fpr3 protein expression. Fpr3 is not only expressed in murine vomeronasal sensory neurons but also in bone marrow cells, the primary source for immune cell renewal, and in mature neutrophils. Consistent with the notion that Fpr3 functions as a pathogen sensor, Fpr3 expression in the immune system is up-regulated after stimulation with a bacterial endotoxin (lipopolysaccharide). These results strongly support a dual role for Fpr3 in both vomeronasal sensory neurons and immune cells. We also identify a large panel of mouse strains with severely altered expression and function of Fpr3, thus establishing the existence of natural Fpr3 knock-out strains. We attribute distinct Fpr3 expression in these strains to the presence or absence of a 12-nucleotide in-frame deletion (Fpr3Δ424-435). In vitro calcium imaging and immunofluorescence analyses demonstrate that the lack of four amino acids leads to an unstable, truncated, and non-functional receptor protein. The genome of at least 19 strains encodes a non-functional Fpr3 variant, whereas at least 13 other strains express an intact receptor. These results provide a foundation for understanding the in vivo function of Fpr3.
Highlights
Social animals benefit from sharing information that promotes fitness and survival [1,2,3]
We previously identified a set of formylated bacterial signal peptides as natural agonists for this receptor [32], and we demonstrated that human FPR3 responds to a similar ligand spectrum [32]
We focused on the detection of the extracellular loops of Fpr3 because these possess the highest sequence divergence among different members of the formyl peptide receptors (Fprs) family
Summary
Peptide-spot Assay for Antibody Characterization—Peptides (15 amino acid residues with an overlap of 10 residues) covering the whole length of Fpr were synthesized on acid-hardened cellulose membranes derivatized with a polyethylene glycol spacer. After rinsing with PBS, staining was obtained by sample incubation for 60 min at room temperature with fluorescence-conjugated secondary antibodies in blocking solution containing Hoechst 33342 (1 g/ml; Hoechst) to counterstain the cell nuclei. In HEK293T cells, Fpr3-ECL2 was stained with a polyclonal goat anti-mouse Alexa Fluor 546 IgG (0.5 g/ml; A-11003; Invitrogen). For VNO and blood samples, we used either a polyclonal goat antimouse Alexa Fluor 488 IgG (2 g/ml; A-11029; Molecular Probes) or a polyclonal donkey anti-mouse Alexa 647 IgG (2 g/ml; A-31571; Invitrogen). Both antibodies were stained with a polyclonal goat anti-mouse Alexa Fluor 546 IgG (0.25 g/ml; A-11003; Invitrogen). All ligands were dissolved in the assay buffer solution C1
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