Role of the Cys 90, Cys 95 and Cys 173 residues in the structure and function of the human platelet-activating factor receptor

Abstract

Platelet-activating factor (PAF) is a potent phospholipid mediator which binds to a specific, high affinity receptor of the G protein-coupled receptor family. In the present report, we show that ligand binding to the PAF receptor is sensitive to the reducing agent dithiothreitol (DTT), suggesting the involvement of disulfide linkages in the proper PAF receptor conformation. Substitutions of Cys 90, Cys 95 and Cys 173 to Ala or Ser demonstrated that these cysteine residues are critical for normal cell surface expression of the PAF receptor protein and ligand binding to the receptor. The Cys 90 and Cys 173 mutant receptors did not display any specific ligand binding, were not expressed on the cell surface but were found in the intracellular compartment. The Cys 95 mutants showed specific binding and were able to stimulate low levels of inositol phosphate (IP) production. These mutants were expressed at low density on the cell surface and showed high expression intracellularly. Our results suggest that the structure and function of the PAF receptor require the conserved Cys 90 and Cys 173 to form a disulfide bond. Moreover, Cys 95 also appears to be necessary, possibly by establishing a disulfide linkage with an as yet unidentified Cys residue. All three residues appear essential for the proper folding and surface expression of the PAF receptor protein.