The binding of leukotriene biosynthesis inhibitors to site-directed mutants of human 5-lipoxygenase-activating protein.
Site-directed mutagenesis was used to develop deletion and point mutants of human 5-lipoxygenase-activating protein (FLAP), which were then expressed in COS-7 cells. Membrane preparations from these cells were analyzed in a radioligand binding assay. Binding of leukotriene biosynthesis inhibitors to FLAP mutants containing deletions of 2 to 6 amino acids within the region from residue 48-61 was undetectable. This finding is consistent with previous studies which suggest that residues amino-terminal to the proposed second transmembrane of FLAP are critical for inhibitor binding. The present study also defines residues of FLAP a) amino-terminal to residue 48, b) between the proposed second and third transmembrane regions and c) in the C-terminal region of the protein which are not involved in inhibitor binding.
Mancini
J A
JA
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Kirkland, Québec, Canada.
Coppolino
M G
MG
Klassen
J H
JH
Charleson
S
S
Vickers
P J
PJ
D058927:5-Lipoxygenase-Activating Proteins; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D015151:Immunoblotting; D007211:Indoles / Q000378:metabolism; D007457:Iodine Radioisotopes; D020024:Leukotriene Antagonists; D015289:Leukotrienes / Q000096:biosynthesis*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics; D011804:Quinolines / Q000378:metabolism; D011869:Radioligand Assay
D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; D002352:Carrier Proteins; D007211:Indoles; D007457:Iodine Radioisotopes; D020024:Leukotriene Antagonists; D015289:Leukotrienes; D008565:Membrane Proteins; D011804:Quinolines; C085966:L 689037; C074616:L 691831