Abstract

5-Lipoxygenase-activating protein (FLAP) is specifically labeled by [125I]L-669,083 and [125I]L-691,678, photoaffinity analogues of two classes of potent leukotriene biosynthesis inhibitors. Because human FLAP contains only a single tryptophan residue at position 72 and two internal methionine residues at positions 89 and 125, we have used reagents that specifically cleave at these residues, in conjunction with antipeptide antisera, to localize the site of attachment of the photoaffinity ligands. Immunoprecipitation of specifically labeled peptide fragments after digestion of photoaffinity-labeled FLAP by iodosobenzoic acid at 72Trp demonstrates that the inhibitors bind to FLAP amino-terminal to this residue. This finding is consistent with similar immunoprecipitation studies after digestion at methionine residues using cyanogen bromide. These findings localize the site of attachment of the inhibitors to a region of FLAP that includes the hydrophilic loop between the proposed first and second transmembrane regions. Based on these findings, site-directed mutagenesis of human FLAP was performed to define key amino acids involved in inhibitor binding. Using a radioligand binding assay, analysis of mutants of human FLAP expressed in COS-7 cells demonstrates that a number of residues in the amino-terminal half of the first hydrophilic loop of the protein can be deleted without significantly affecting inhibitor binding. In contrast, no inhibitor binding was detectable with mutants in which amino acid residues in the carboxyl-terminal half of this loop were deleted. Furthermore, a point mutation of 62Asp to asparagine results in a mutant with dramatically reduced affinity for inhibitors. This loss of affinity was not displayed by a mutant in which 62Asp was mutated to a glutamate residue, suggesting that a negative charge associated with residue 62 may be critical for inhibitor binding. The roles that amino acid residues in the carboxyl-terminal half of the first hydrophilic loop of FLAP may play in the binding of leukotriene biosynthesis inhibitors are currently under investigation.