Functional Structure Analysis of Ion-channel Oligomer and Production of regulating Peptides.

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Human formyl peptide receptor (FPR) mediates a number of important host defense functions. Although studies have been made on ligand binding site of FPR, FPR dynamic behaviors on cell surface is unknown. Recently, peptides derived from transmembrane (TM) domain of GPCRs were shown to disrupt dimer formation of receptors and results in specific regulation of receptor function. To reveal the function of FPR TM domains, hFPRTM peptides derived from human FPR were synthesized, and their biological activities were evaluated on human neutrophils. TM peptides were synthesized through a stepwise solid phase method using Fmoc amino acid on low substituted resin. The purifications of crude TM peptides were carried out by preparative RP-HPLC using water/2-propanol/formic acid system. The homogeneities and structures of synthetic peptides were verified by analytical RP-HPLC and MALDI-TOF MS. Synthetic hFPRTM peptides did not exhibit agonistic or antagonistic activities on superoxide anion production in human neutrophils. However, human neutrophils treated with hFPRTM4 produced 4-folds superoxide anion compared with untreated cells. Short peptides fragments from fourth TM region of human FPR did not enhance superoxide anion production, which suggest that hFPRTM4 did not behave as a ligand. CD and fluorescence spectra suggested that hFPRTM peptides inserted into the membrane and interacted with TM domain of membrane proteins. Addition of hFPRTM4 increased intracellular calcium concentration, which meant the peptide activate some membrane protein on cell surface. Present study suggests that the fourth TM domain of FPR has novel function related to priming effect.