Single nucleotide polymorphism of human platelet-activating factor receptor impairs G-protein activation.

Koichiro Asano,Satoshi Ishii,Takao Shimizu,Kazuhiro Yamaguchi,Kouichi Fukunaga,Tetsuya Shiomi,Takehiko Yokomizo

doi:10.1074/jbc.m108288200

Abstract

Various proinflammatory and vasoactive actions of platelet-activating factor (PAF) are mediated through a specific G-protein-coupled PAF receptor (PAFR). We identified a novel DNA variant in the human PAFR gene, which substitutes an aspartic acid for an alanine residue at position 224 (A224D) in the putative third cytoplasmic loop. This mutation was observed in a Japanese population at an allele frequency of 7.8%. To delineate the functional consequences of this structural alteration, Chinese hamster ovary cells were stably transfected with constructs encoding either wild-type or A224D mutated PAFR. No significant difference was observed in the expression level of the receptor or the affinity to PAF or to an antagonist, WEB2086, between the cells transfected with wild-type and mutant PAFR. Chinese hamster ovary cells expressing A224D mutant PAFR displayed partial but significant reduction of PAF-induced intracellular signals such as calcium mobilization, inositol phosphate production, inhibition of adenylyl cyclase, and chemotaxis. These findings suggest that this variant receptor produced by a naturally occurring mutation exhibits impaired coupling to G-proteins and may be a basis for interindividual variation in PAF-related physiological responses, disease predisposition or phenotypes, and drug responsiveness.

Highlights

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3phosphocholine) receptor (PAFR)1 with seven-transmembrane domain structure belongs to the G-protein-coupled receptor superfamily [1, 2]
Intracellular Calcium Mobilization—Because the amino acid substitution A224D occurs in the putative third cytoplasmic loop that could be essential for the interaction with G-proteins [11], we examined the effect of this mutation on the intracellular signal transduction
We analyzed the human PAFR gene in search for the host genetic factors that modify the biological phenotypes related to platelet-activating factor (PAF)-PAFR system and identified a single amino acid substitution (A224D) in the third cytoplasmic loop of human PAFR

Summary

Introduction

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3phosphocholine) receptor (PAFR) with seven-transmembrane domain structure belongs to the G-protein-coupled receptor superfamily [1, 2]. Responsiveness to PAF in vivo demonstrates a significant intersubject variation in human subjects and mice. Brzustowicz et al [6] examined PAFevoked calcium transients in immortalized human B lymphocytes and demonstrated that there is a substantial intersubject difference, suggesting that the interindividual variation of the responses to PAF occurs at the level of PAFR or its downstream signaling. Such variation can be explained by the genetic polymorphisms in the receptor itself, its cognate G-proteins, or downstream intracellular targets. We analyzed the ligand binding and signal transduction properties of the mutant receptor by expressing it in mammalian cell lines

Methods

Results

Conclusion