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J Biol Chem, Vol. 275, Issue 19, 14281-14286, May 12, 2000

Characterization of a Novel Sphingosine 1-Phosphate Receptor, Edg-8*

Dong-Soon ImDagger , Christopher E. HeiseDagger §, Nicolas Ancellin, Brian F. O'Dowd||, Gan-ju Shei**, Robert P. HeavensDagger Dagger , Michael R. RigbyDagger Dagger , Timothy Hla§§, Suzanne Mandala**, George McAllister**, Susan R. George||, and Kevin R. LynchDagger ¶¶

From the Dagger  Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, the  Center for Vascular Biology and Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06030, the || Center for Addiction and Mental Health and University of Toronto, Toronto, Ontario M5S 2S1, Canada, the ** Department of Infectious Disease, Merck Research Laboratories, Rahway, New Jersey 07065, and Dagger Dagger  Merck, Sharpe & Dohme, Neuroscience Research Center, Harlow CM20 2QR, United Kingdom

Three G protein-coupled receptors (Edg-1, Edg-3, and Edg-5) for the lysolipid phosphoric acid mediator sphingosine 1-phosphate have been described by molecular cloning. Using a similar sequence that we found in the expressed sequence tag data base, we cloned and characterized of a fourth, high affinity, rat brain sphingosine 1-phosphate receptor, Edg-8. When HEK293T cells were co-transfected with Edg-8 and G protein DNAs, prepared membranes showed sphingosine 1- phosphate-dependent increases in [35S]guanosine 5'-(3-O-thio)triphosphate binding with an EC50 of 90 nM. In a rat hepatoma Rh7777 cell line that exhibits modest endogenous responses to sphingosine 1-phosphate, this lipid mediator inhibited forskolin-driven rises in cAMP by greater than 90% when the cells were transfected with Edg-8 DNA (IC50 0.7 nM). This response is blocked fully by prior treatment of cultures with pertussis toxin, thus implicating signaling through Gi/oalpha proteins. Furthermore, Xenopus oocytes exhibit a calcium response to sphingosine 1-phosphate after injection of Edg-8 mRNA, but only when oocytes are co-injected with chimeric Gq/ialpha protein mRNA. Membranes from HEK293T and Rh7777 cell cultures expressing Edg-8 exhibited high affinity (KD = 2 nM) binding for radiolabeled sphingosine 1-phosphate. Rat Edg-8 RNA is expressed in spleen and throughout adult rat brain where in situ hybridization revealed it to be associated with white matter. Together our data demonstrate that Edg-8 is a high affinity sphingosine 1-phosphate receptor that couples to Gi/oalpha proteins and is expressed predominantly by oligodendrocytes and/or fibrous astrocytes in the rat brain.

* This work was supported in part by Research Grants R01 GM52722 (to K. R. L.) and R01 DK4569 (to T. H.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF233649.

§ Supported by National Research Service Award Traineeship T32 GM07055.

§§ Supported by an Established Investigator award from the American Heart Association.

¶¶ To whom correspondence should be addressed: Dept. Pharmacology, Rm. 5221, University of Virginia School of Medicine, 1300 Jefferson Park Ave., Charlottesville, VA 22908. Tel.: 804-924-2840; Fax: 804-982-3878; E-mail: KRL2Z@virginia.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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