Role of the Cys90, Cys95 and Cys173 residues in the structure and function of the human platelet-activating factor receptor.
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.
Le Gouill
C
C
Department of Pediatrics, Faculty of Medicine, University of Sherbrooke, Qué., Canada.
Parent
J L
JL
Rola-Pleszczynski
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M
Stanková
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001381:Azepines / Q000378:metabolism; D001667:Binding, Competitive; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D003545:Cysteine; D017931:DNA Primers; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D010972:Platelet Activating Factor / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014230:Triazoles / Q000378:metabolism
D001381:Azepines; D017931:DNA Primers; D007295:Inositol Phosphates; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D016271:Proto-Oncogene Proteins c-myc; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D014230:Triazoles; C039351:platelet activating factor receptor; C052518:WEB 2086; D003545:Cysteine; D004229:Dithiothreitol