Characterization of a novel sphingosine 1-phosphate receptor, Edg-8

Abstract

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 G(i/o)α 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 G(q/i)α protein mRNA. Membranes from HEK293T and Rh7777 cell cultures expressing Edg-8 exhibited high affinity (K(D) = 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 G(i/o)α proteins and is expressed predominantly by oligodendrocytes and/or fibrous astrocytes in the rat brain.