Biochemical and Biophysical Research Communications
N-Formylated humanin activates both formyl peptide receptor-like 1 and 2☆
Section snippets
Experimental procedures
Peptides. All peptides were purchased from Peptide Institute (Osaka, Japan).
Preparation of CHO cells expressing GPCRs. The entire coding regions of hFPR1, hFPRL1 or hFPRL2 cDNAs were inserted downstream of the SRα promoter in an expression vector pAKKO-111H [30]. To express the fusion protein of hFPRL1 and GFP, a vector plasmid was constructed by inserting a fused DNA, in which hFPRL1- and GFP-coding regions were tandemly connected in-frame, into pAKKO-111H. These expression plasmids were
Identification of HN as a ligand for hFPRL1
We prepared CHO-hFPRL1-GFP cells to screen for ligands of hFPRL1. GFP was used as a marker to ascertain the expression of hFPRL1. We screened more than 1000 compounds by measuring the suppression of forskolin-induced cAMP production in CHO-hFPRL1-GFP, and detected a specific response to HN and HNG at 0.4 μM (data not shown). In the absence of a ligand, the hFPRL1-GFP was typically localized at the plasma membrane (Fig. 1A). In contrast, in the presence of HNG, the hFPRL1-GFP was internalized
Discussion
In this paper, we have demonstrated that HN and its related peptides act as specific ligands for FPRL1 and 2. Although it has been recently reported that HN uses FPRL1 as a functional receptor [35], we believe this is the first report demonstrating that HN acts as a ligand for not only FPRL1 but also FPRL2. Furthermore, until now there has been no report demonstrating direct interaction between HN and FPRL1. Through our binding assays using [125I]-W peptide, we have shown here the direct
Acknowledgments
We thank Drs. M. Fujino, T. Soda, K. Okonogi, Y. Ito, F. Itoh, and K. Fukatsu for their helpful discussions and collaboration.
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Abbreviations: HN, humanin; FPRL1, formyl peptide receptor-like 1; FPRL2, formyl peptide receptor-like 2; FPR1, formyl peptide receptor 1; CHO, Chinese hamster ovary; fHN, N-formylated humanin; EC50, half-maximal response; GPCRs, G-protein-coupled receptors; GFP, green fluorescence protein; AD, Alzheimer’s disease; Aβ, β-amyloid peptide; IC50, 50% inhibition of specific binding; DMEM, Dulbecco’s modified minimum essential medium; FAM, 6-carboxyfluorescein; TAMRA, 6-carboxy-tetramethylrhodamine; HNG, [Gly14]-humanin; PMNs, polymorphonuclear leukocytes.