ROLE OF THE CYS(90), CYS(95) AND CYS(173) RESIDUES IN THE STRUCTURE AND FUNCTION OF THE HUMAN PLATELET-ACTIVATING-FACTOR RECEPTOR

Platelet-activating factor (PAF) is a potent phospholipid mediator whi ch binds to a specific, high affinity receptor of the G protein-couple d receptor family, In the present report, me 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(1 73) to Ala or Ser demonstrated that these cysteine residues are critic al for normal cell surface expression of the PAF receptor protein and ligand binding to the receptor, The Cys(90) and Cys(173) mutant recept ors did not display any specific ligand binding, were not expressed on the cell surface but mere found in the intracellular compartment, The Cys(95) mutants showed specific binding and were able to stimulate lo w levels of inositol phosphate (IF) production, These mutants were exp ressed at low density on the cell surface and showed high expression i ntracellularly. 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, possi bly by establishing a disulfide linkage with an as yet unidentified Cy s residue, All three residues appear essential for the proper folding and surface expression of the PAF receptor protein.