title: Identification of transmembrane domain residues determinant in the structure-function relationship of the human platelet-activating factor receptor by site-directed mutagenesis.
abstract: Platelet-activating factor (PAF) is a potent phospholipid mediator that produces a wide range of biological responses. The PAF receptor is a member of the seven-transmembrane GTP-binding regulatory protein-coupled receptor superfamily. This receptor binds PAF with high affinity and couples to multiple signaling pathways, leading to physiological responses that can be inhibited by various structurally distinct PAF antagonists. We have used site-directed mutagenesis and functional expression studies to examine the role of the Phe97 and Phe98 residues located in the third transmembrane helix and Asn285 and Asp289 of the seventh transmembrane helix in ligand binding and activation of the human PAF receptor in transiently transfected COS-7 cells. The double mutant FFGG (Phe97 and Phe98 mutated into Gly residues) showed a 3-4-fold decrease in affinity for PAF, but not for the specific antagonist WEB2086, when compared with the wild-type (WT) receptor. The FFGG mutant receptor, however, displayed normal agonist activation, suggesting that these two adjacent Phe residues maintain the native PAF receptor conformation rather than interacting with the ligand. On the other hand, substitution of Ala for Asp289 increased the receptor affinity for PAF but abolished PAF-dependent inositol phosphate accumulation; it did not affect WEB2086 binding. Substitution of Asn for Asp289, however, resulted in a mutant receptor with normal binding and activation characteristics. When Asn285 was mutated to Ala, the resulting receptor was undistinguishable from the WT receptor. Surprisingly, substitution of Ile for Asn285 led to a loss of ligand binding despite normal cell surface expression levels of this mutant, as verified by flow cytometric analysis. Our data suggest that residues 285 and 289 are determinant in the structure and activation of the PAF receptor but not in direct ligand binding, as had been recently proposed in a PAF receptor molecular model.
authors.affiliation: Immunology Division, Department of Pediatrics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
authors.forename: J L
authors.initials: JL
authors.lastname: Parent
authors.forename: C L
authors.initials: CL
authors.lastname: Gouill
authors.forename: E
authors.initials: E
authors.lastname: Escher
authors.forename: M
authors.initials: M
authors.lastname: Rola-Pleszczynski
authors.forename: J
authors.initials: J
authors.lastname: Staková
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001381:Azepines / Q000494:pharmacology; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D010972:Platelet Activating Factor / Q000378:metabolism*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D013329:Structure-Activity Relationship; D014162:Transfection; D014230:Triazoles / Q000494:pharmacology
chemical_list: D001381:Azepines; D007295:Inositol Phosphates; D010972:Platelet Activating Factor; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D016271:Proto-Oncogene Proteins c-myc; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D014230:Triazoles; C039351:platelet activating factor receptor; C052518:WEB 2086; D001224:Aspartic Acid; D010649:Phenylalanine; D001216:Asparagine