Lipid Rafts Facilitate the Interaction of PECAM-1 with the Glycoprotein VI-FcR γ-Chain Complex in Human Platelets

Fiona A Lee,Marjolijn Van Lier,Ingrid A.M Relou,Loraine Foley,Jan-Willem N Akkerman,Harry F.G Heijnen,Richard W Farndale

doi:10.1074/jbc.m607930200

Abstract

Glycoprotein (GP) VI, the main signaling receptor for collagen on platelets, is expressed in complex with the FcR gamma-chain. The latter contains an immunoreceptor tyrosine-based activation motif, which becomes phosphorylated, initiating a signaling cascade leading to the rapid activation and aggregation of platelets. Previous studies have shown that signaling by immunoreceptor tyrosine-based activation motif-containing receptors is counteracted by signals from receptors with immunoreceptor tyrosine-based inhibitory motifs. Here we show, by immunoprecipitation, that the GPVI-FcR gamma-chain complex associates with the immunoreceptor tyrosine-based inhibitory motif-containing receptor, PECAM-1. In platelets stimulated with collagen-related peptide (CRP-XL), tyrosine phosphorylation of PECAM-1 precedes that of the FcR gamma-chain, implying direct regulation of the former. The GPVI-FcR gamma-chain complex and PECAM-1 were present in both lipid raft and soluble fractions in human platelets; this distribution was unaltered by activation with CRP-XL. Their association occurred in lipid rafts and was lost after lipid raft depletion using methyl-beta-cyclodextrin. We propose that lipid raft clustering facilitates the interaction of PECAM-1 with the GPVI-FcR gamma-chain complex, leading to the down-regulation of the latter.

Highlights

The discovery of specialized microdomains in the plasma membrane known as lipid rafts or detergent-resistant membranes has opened a new field of research, providing insight into the role of the plasma membrane in signal transduction [1]
We show by immunoprecipitation that there is a physical association of PECAM-1 with the GPVI-FcR ␥-chain complex and that this is facilitated by lipid rafts
Much less is known about the immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing inhibitory receptors that can modulate these signals, and recent evidence suggests a role for ITIMs in mediating platelet activation [28]

Summary

Introduction

The discovery of specialized microdomains in the plasma membrane known as lipid rafts or detergent-resistant membranes has opened a new field of research, providing insight into the role of the plasma membrane in signal transduction [1]. We show by immunoprecipitation that there is a physical association of PECAM-1 with the GPVI-FcR ␥-chain complex and that this is facilitated by lipid rafts. Whole cell lysates and immunoprecipitates of GPVI, the FcR ␥-chain, or PECAM-1 were separated by SDSPAGE, and associated proteins were immunodetected.

Results

Conclusion