The Synthetic Peptide Trp-Lys-Tyr-Met-Val-Met-NH2 Specifically Activates Neutrophils through FPRL1/Lipoxin A4 Receptors and Is an Agonist for the Orphan Monocyte-expressed Chemoattractant Receptor FPRL2

Abstract

The extravasation of leukocytes from the peripheral blood stream to inflammatory sites is a key feature in the innate immune response to infection (1). Different chemoattractants (e.g. N-formylated peptides, C5a, interleukin-8, leukotriene B 4 , and platelet-activating factor) and chemokines induce leukocyte infiltration and activation through binding to G proteincoupled seven-transmembrane cell-surface receptors (2, 3). The chemoattractant-mediated dissociation of G␣ i2 from the G␤␥ subunit complex results in the activation of several downstream signaling effector enzymes that promote intracellular calcium mobilization, modifications in the metabolism of phosphoinositides, and activation of mitogen-activated protein kinases (4). The integration by the cell of the different chemoattractant-activated signaling pathways results in directed cell migration, recruitment of new receptors from the granules to the cell surface, release of proteolytic enzymes, production of large amounts of superoxide by the neutrophil NADPH oxidase, and increased gene transcription (5-8). The extent of the cellular response is dependent on the identity of the agonist and on the level of expression and desensitization of the receptors involved in the activation process (9).Two synthetic hexapeptides, Trp-Lys-Tyr-Met-Val-Met-NH 2 (WKYMVM)andTrp-Lys-Tyr-Met-Val-D-Met-NH 2 (WKYMVm), that stimulate phosphoinositide hydrolysis in myeloid cells were identified by screening a peptide library (10, 11). The D-methionine-containing hexapeptide (WKYMVm) was found to be a very potent activator of several leukocyte effector functions such as chemotaxis, mobilization of complement receptor-3, and activation of the NADPH oxidase (11). The peptide WKYMVm activates neutrophils through both the N-formyl peptide receptor (FPR) 1 and FPRL1 (N-formyl peptide receptor-like-1) (12, 13). The latter was originally cloned from human phagocytes by low-stringency hybridization of a cDNA library with the FPR cDNA sequence, and it was initially defined as an orphan receptor (14 -16). FPRL1 was later referred to as the LXA 4 receptor since it was shown to bind lipoxin A 4 with high affinity (17). In addition, several different peptides/proteins have been reported to stimulate this receptor. These include a leucine zipper-like domain of the HIV-1

Human mitochondria-derivedN-formylated peptides are novel agonists equally active on FPR and FPRL1, whileListeria monocytogenes-derived peptides preferentially activate FPR

“…Each one of these receptors was stably expressed in HL-60 cells, a cell line of myeloid origin that does not express these receptors when undifferentiated [14]. The transfected cells were used to investigate the receptor specificity of the peptides.…”

“…The construction of the expression plasmids and the stable expression of FPRL1 and FPRL2 in undifferentiated HL-60 cells have been previously described [14,19]. Both cell lines gave an intracellular calcium response peaking at 400-600 nM after stimulation with 1 lM of the hexapeptide WKYMVm.…”

“…The chemotaxis assay was performed as previously described [14]. In brief, undifferentiated HL-60 cells stably expressing either FPRL1 or FPRL2 were centrifuged, resuspended in fresh culture medium (10 6 cells/mL), and incubated overnight with 10 lg/mL 1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethylindolcarbocyanine perchlorate (Molecular probes, Eugene, OR).…”

“…Recently, FPRL1 has emerged as a promiscuous receptor, also activated by the pathogenderived peptide Hp (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) from Helicobacter pylori, by host-derived proteins and peptides such as Ac1-25 from annexin 1, cathelicidin LL37, a fragment of the urokinase receptor, the serum amyloid A, the amyloid b peptide (Ab42), the prion protein fragment PrP(106-126), the neuroprotective peptide humanin, and by different synthetic peptides including the hexapeptide Trp-LysTyr-Met-Val-D-Met-NH 2 (WKYMVm) [11,12]. FPRL2 does not respond to fMLF [13], but instead is activated by some non-formylated chemotactic peptides identified as agonists for FPRL1 [14,15]. Recently, an acetylated Nterminal peptide derived from the human heme-binding protein has been identified as a ligand specific for FPRL2 [16].…”

An Annexin 1 N-Terminal Peptide Activates Leukocytes by Triggering Different Members of the Formyl Peptide Receptor Family

“…In contrast to FPR and FPRL1, FPRL2 is an orphan Institute for Medical Biochemistry, Center for Molecular Biology of Inflammation, Münster, Germany receptor and does not bind fMLP. The only FPRL2 ligand identified so far is the unnatural synthetic peptide WKYMVM (24).…”