Platelet-activating factor (PAF) is a potent phospholipid mediator with diverse physiological actions on different types of cells and tissues. In spite of its highly hydrophobic structure, PAF binds to a specific cell surface receptor, which belongs to a G p rotein-coupled, seven transrnembrane receptor superfamily. To investigate ligand binding sites in a PAF receptor, we chose a mutagenesis approach. From previous extensive study of structure-activity relationships, intermolecular polarity in the alkyl-ether bond at sn-1 position, the acetyl moiety at sn-2 position, and the phosphorylcholine moiety at sn-3 position seems to be crucial for its receptor binding. We have exchanged independently all 24 polar amino acids, corresponding to the putative transmembrane domains of a guinea-pig PAF receptor, to non-polar alanine. Mutated receptors were expressed transiently in COS-7 cells, and PAF binding activities were measured and normalized by receptor expression levels. Among them, some mutants displayed altered affinity to its ligand PAF comparing with a wild type receptor. PAF scarcely bound to some mutants which diminished polar (especially charged) amino acids in several transmembrane domains. In contrast, PAF bound preferably to 2nd and 3rd transmembrane mutants. Binding affinities of a receptor antagonist (WEB2086) were indistinguishable between the wild type and these mutants. Next, we constructed 6 kinds of CHO stable transformants expressing representative mutants. These CHO transformants had almost same characteristics in ligand binding as those in equivalent transfected COS-7 cells. In conclusion, we have found that polar amino acid exchanges in transmembrane regions generate PAF receptor mutants with altered affinities to their ligand.
|Publication status||Published - 1996|
ASJC Scopus subject areas
- Molecular Biology