Sphingosine 1-Phosphate-induced Cell Rounding and Neurite Retraction Are Mediated by the G Protein-coupled Receptor H218

James R Van Brocklyn,Zhenxing Tu,Lisa C Edsall,Richard R Schmidt,Sarah Spiegel

doi:10.1074/jbc.274.8.4626

Abstract

Sphingosine 1-phosphate (SPP) is a lipid second messenger that also acts as a first messenger through the G protein-coupled receptor Edg-1. Here we show that SPP also binds to the related receptors H218 and Edg-3 with high affinity and specificity. SPP and sphinganine 1-phosphate bind to these receptors, whereas neither sphingosylphosphorylcholine nor lysophosphatidic acid compete with SPP for binding to either receptor. Transfection of HEK293 cells with H218 or edg-3, but not edg-1, induces rounded cell morphology in the presence of serum, which contains high levels of SPP. SPP treatment of cells overexpressing H218 cultured in delipidated serum causes cell rounding. A similar but less dramatic effect was observed in cells overexpressing Edg-3 but not with Edg-1. Cell rounding was correlated with apoptotic cell death, probably as a result of loss of attachment. Nerve growth factor-induced neuritogenesis in PC12 cells was inhibited by overexpression of H218 and to a lesser extent Edg-3. SPP treatment rapidly enhanced neurite retraction in PC12 cells overexpressing Edg-1, Edg-3, or H218. Thus, H218, and possibly Edg-3, may be the cell surface receptors responsible for cell rounding and neurite retraction induced by SPP. Moreover, the identification of these two additional SPP receptors indicates that a family of highly specific receptors exists that mediate different responses to SPP.

Highlights

The sphingolipid metabolite sphingosine 1-phosphate (SPP)1 is emerging as a member of a new class of lipid second messengers [1, 2]
Sphingosine 1-Phosphate Binds to H218 and Edg-3—Quantitative binding studies with lysolipid agonists such as SPP and lysophosphatidic acid (LPA) have been hampered by the lipophilic nature of the ligand, resulting in very high levels of nonspecific binding
To determine if the Edg-3 and H218 G protein-coupled receptors, which share 48 and 45% homology, respectively, with endothelial differentiation gene-1 (Edg-1), were capable of binding SPP, HEK293 cells were transiently transfected with pcDNA3 expression plasmids containing H218 or edg-3 open reading frames with c-myc tags fused in frame at the N terminus

Summary

Introduction

The sphingolipid metabolite sphingosine 1-phosphate (SPP)1 is emerging as a member of a new class of lipid second messengers [1, 2]. To determine if the Edg-3 and H218 G protein-coupled receptors, which share 48 and 45% homology, respectively, with Edg-1, were capable of binding SPP, HEK293 cells were transiently transfected with pcDNA3 expression plasmids containing H218 or edg-3 open reading frames with c-myc tags fused in frame at the N terminus. H218- and Edg-3-expressing HEK293 cells display dramatically increased specific binding of [32P]SPP in comparison with untransfected and vector-transfected cells (Fig. 1B).

Results

Conclusion