Le Gouill C, Parent J L, Rola-Pleszczynski M, Stanková J
Department of Pediatrics, Faculty of Medicine, University of Sherbrooke, Qué., Canada.
FEBS Lett. 1997 Feb 3;402(2-3):203-8. doi: 10.1016/s0014-5793(96)01531-1.
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 Cys90, Cys95 and Cys173 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 Cys90 and Cys173 mutant receptors did not display any specific ligand binding, were not expressed on the cell surface but were found in the intracellular compartment. The Cys95 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 Cys90 and Cys173 to form a disulfide bond. Moreover, Cys95 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.
血小板活化因子（PAF）是一种强效磷脂介质，它与G蛋白偶联受体家族的一种特异性、高亲和力受体结合。在本报告中，我们表明配体与PAF受体的结合对还原剂二硫苏糖醇（DTT）敏感，这表明二硫键参与了PAF受体的正确构象形成。将半胱氨酸90（Cys90）、半胱氨酸95（Cys95）和半胱氨酸173（Cys173）替换为丙氨酸（Ala）或丝氨酸（Ser）表明，这些半胱氨酸残基对于PAF受体蛋白在细胞表面的正常表达以及配体与受体的结合至关重要。Cys90和Cys173突变体受体未表现出任何特异性配体结合，未在细胞表面表达，但存在于细胞内区室中。Cys95突变体表现出特异性结合，并能够刺激产生低水平的肌醇磷酸（IP）。这些突变体在细胞表面以低密度表达，在细胞内表现出高表达。我们的结果表明，PAF受体的结构和功能需要保守的Cys90和Cys173形成二硫键。此外，Cys95似乎也是必需的，可能是通过与一个尚未确定的半胱氨酸残基建立二硫键。所有这三个残基对于PAF受体蛋白正确折叠和表面表达似乎都是必不可少的。
