IDENTIFICATION OF TRANSMEMBRANE DOMAIN RESIDUES DETERMINANT IN THE STRUCTURE-FUNCTION RELATIONSHIP OF THE HUMAN PLATELET-ACTIVATING-FACTOR RECEPTOR BY SITE-DIRECTED MUTAGENESIS

Citation
Jl. Parent et al., IDENTIFICATION OF TRANSMEMBRANE DOMAIN RESIDUES DETERMINANT IN THE STRUCTURE-FUNCTION RELATIONSHIP OF THE HUMAN PLATELET-ACTIVATING-FACTOR RECEPTOR BY SITE-DIRECTED MUTAGENESIS, The Journal of biological chemistry, 271(38), 1996, pp. 23298-23303
Citations number
35
Categorie Soggetti
Biology
ISSN journal
00219258
Volume
271
Issue
38
Year of publication
1996
Pages
23298 - 23303
Database
ISI
SICI code
0021-9258(1996)271:38<23298:IOTDRD>2.0.ZU;2-G
Abstract
Platelet-activating factor (PAF) is a potent phospholipid mediator tha t produces a wide range of biological responses. The PAF receptor is a member of the seven-transmembrane GTP binding regulatory protein-coup led receptor superfamily. This receptor binds PAF with high affinity a nd couples to multiple signaling pathways, leading to physiological re sponses that can be inhibited by various structurally distinct PAF ant agonists. We have used site-directed mutagenesis and functional expres sion studies to examine the role of the Phe(97) and Phe(98) residues l ocated in the third transmembrane helix and Asn(285) and Asp(289) of t he seventh transmembrane helix in ligand binding and activation of the human PAF receptor in transiently transfected COS-7 cells. The double mutant FFGG (Phe(97) and Phe(98) mutated into Gly residues) showed a 3-4-fold decrease in affinity for PAF, but not for the specific antago nist WEB2086, when compared with the wild-type (WT) receptor. The FFGG mutant receptor, however, displayed normal agonist activation, sugges ting that these two adjacent Phe residues maintain the native PAF rece ptor conformation rather than interacting with the ligand. On the othe r hand, substitution of Ala for Asp(289) increased the receptor affini ty for PAF but abolished PAF-dependent inositol phosphate accumulation ; it did not affect WEB2086 binding. Substitution of Asn for Asp(289), however, resulted in a mutant receptor with normal binding and activa tion characteristics. When Asn(285) was mutated to Ala, the resulting receptor was undistinguishable from the WT receptor. Surprisingly, sub stitution of Ile for Asn(285) led to a loss of ligand binding despite normal cell surface expression levels of this mutant, as verified by f low cytometric analysis. Our data suggest that residues 285 and 289 ar e determinant in the structure and activation of the PAF receptor but not in direct ligand binding, as had been recently proposed in a PAF r eceptor molecular model.