N-Formylated humanin activates both formyl peptide receptor-like 1 and 2

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Abstract

We have discovered that humanin (HN) acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2). This discovery was based on our finding that HN suppressed forskolin-induced cAMP production in Chinese hamster ovary (CHO) cells expressing human FPRL1 (CHO-hFPRL1) or human FPRL2 (CHO-hFPRL2). In addition, we found that N-formylated HN (fHN) performed more potently as a ligand for FPRL1 than HN: in CHO-hFPRL1 cells, the effective concentration for the half-maximal response (EC50) value of HN was 3.5 nM, while that of fHN was 0.012 nM. We demonstrated by binding experiments using [125I]-W peptide that HN and fHN directly interacted with hFPRL1 on the membrane. In addition, we found that HN and fHN showed strong chemotactic activity for CHO-hFPRL1 and CHO-hFPRL2 cells. HN is known to have a protective effect against neuronal cell death. Our findings contribute to the understanding of the mechanism behind HN’s function.

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.

References (36)

  • X. Deng et al.

    A synthetic peptide derived from human immunodeficiency virus type 1 gp120 downregulates the expression and function of chemokine receptors CCR5 and CXCR4 in monocytes by activating the 7-transmembrane G-protein-coupled receptor FPRL1/LXA4R

    Blood

    (1999)
  • Y. Le et al.

    Formyl-peptide receptors revisited

    Trends Immunol.

    (2002)
  • Y. Hashimoto et al.

    Mechanisms of neuroprotection by a novel rescue factor humanin from Swedish mutant amyloid precursor protein

    Biochem. Biophys. Res. Commun.

    (2001)
  • S. Hinuma et al.

    Molecular cloning and functional expression of a human thyrotropin-releasing hormone (TRH) receptor gene

    Biochem. Biophys. Acta

    (1994)
  • S. Fukusumi et al.

    Characteristics and distribution of endogenous RFamide-related peptide-1

    Biochem. Biophys. Acta

    (2001)
  • R. Fujii et al.

    Tissue distribution of prolactin-releasing peptide (PrRP) and its receptor

    Regul. Pept.

    (1999)
  • S. Hinuma et al.

    A prolactin-releasing peptide in the brain

    Nature

    (1998)
  • S. Hinuma et al.

    The quest for novel bioactive peptides utilizing orphan seven-transmembrane-domain receptors

    J. Mol. Med.

    (1999)
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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.

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