Sphingosine-1-phosphate is a high-affinity ligand for the G protein-coupled receptor GPR6 from mouse and induces intracellular Ca2+ release by activating the sphingosine-kinase pathway

Abstract EDG-6 is a recently cloned member of the endothelial differentiation gene (EDG) G protein-coupled receptor family that is expressed in lymphoid and hematopoietic tissue and in the lung. Homology of EDG-6 to the known sphingosine-1-phosphate (SPP) receptors EDG-1, EDG-3, and EDG-5 and lysophosphatidic acid (LPA) receptors EDG-2 and EDG-4 suggested that its ligand may be a lysophospholipid or lysosphingolipid. We examined the binding of [32P]SPP to HEK293 cells, transiently transfected with cDNA encoding EDG-6. Binding of [32P]SPP was saturable, demonstrating high affinity (KD = 63 nmol/L). Binding was also specific for SPP, as only unlabeled SPP and sphinganine-1-phosphate, which lacks the trans double bond at the 4 position, potently displaced radiolabeled SPP. LPA did not compete for binding of SPP at any concentration tested, whereas sphingosylphosphorylcholine competed for binding to EDG-6, but only at very high concentrations. In addition, SPP activated extracellular signal-regulated kinase (Erk) in EDG-6 transfected cells in a pertussis toxin-sensitive manner. These results indicate that EDG-6 is a high affinity receptor for SPP, which couples to a Gi/o protein, resulting in the activation of growth-related signaling pathways.

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Sphingosine-1-phosphate is a ligand for the G protein-coupled receptor EDG-6

Blood ◽

10.1182/blood.v95.8.2624.008k33_2624_2629 ◽

2000 ◽

Vol 95 (8) ◽

pp. 2624-2629

Author(s):

James R. Van Brocklyn ◽

Markus H. Gräler ◽

Günter Bernhardt ◽

John P. Hobson ◽

Martin Lipp ◽

...

Keyword(s):

G Protein ◽

Hek293 Cells ◽

Hematopoietic Tissue ◽

G Protein Coupled Receptor ◽

High Affinity ◽

Extracellular Signal ◽

Sphingosine 1 Phosphate ◽

High Affinity Receptor ◽

High Concentrations ◽

G Protein Coupled

EDG-6 is a recently cloned member of the endothelial differentiation gene (EDG) G protein-coupled receptor family that is expressed in lymphoid and hematopoietic tissue and in the lung. Homology of EDG-6 to the known sphingosine-1-phosphate (SPP) receptors EDG-1, EDG-3, and EDG-5 and lysophosphatidic acid (LPA) receptors EDG-2 and EDG-4 suggested that its ligand may be a lysophospholipid or lysosphingolipid. We examined the binding of [32P]SPP to HEK293 cells, transiently transfected with cDNA encoding EDG-6. Binding of [32P]SPP was saturable, demonstrating high affinity (KD = 63 nmol/L). Binding was also specific for SPP, as only unlabeled SPP and sphinganine-1-phosphate, which lacks the trans double bond at the 4 position, potently displaced radiolabeled SPP. LPA did not compete for binding of SPP at any concentration tested, whereas sphingosylphosphorylcholine competed for binding to EDG-6, but only at very high concentrations. In addition, SPP activated extracellular signal-regulated kinase (Erk) in EDG-6 transfected cells in a pertussis toxin-sensitive manner. These results indicate that EDG-6 is a high affinity receptor for SPP, which couples to a Gi/o protein, resulting in the activation of growth-related signaling pathways.

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