Characterization of the human and mouse sphingosine 1-phosphate receptor, S1P(5) (Edg-8): Structure-activity relationship of sphingosine1-phosphate receptors

Five G protein-coupled receptors (S1P(1)/Edg-1, S1P(3)/Edg-3, S1P(2)/Edg-5, S1P(4)/Edg-6, and S1P(5)/Edg-8) for the intercellular lipid mediator sphin gosine 1-phosphate have been cloned and characterized. We found human and m ouse sequences closely related to rat S1P(5) (97% identical amino acids) an d report now the characterization of the human and mouse S1P(5) gene produc ts as encoding sphingosine 1-phosphate receptors. When HEK293T cells were c otransfected with S1P(5) and G protein DNAs, prepared membranes showed sphi ngosine 1-phosphate concentration-dependent increases in [gamma-S-35]GTP bi nding (EC50 = 12.7 nM). The lipid mediator inhibited forskolin-driven rises in cAMP by greater than 80% after introduction of the mouse or human S1P(5 ) DNAs into rat hepatoma RH7777 cells (IC50 = 0.22 nM). This response is bl ocked fully by prior treatment of cultures with pertussis toxin, thus impli cating signaling through G(i/o)alpha proteins. Northern blot analysis showe d high expression of human S1P(5) mRNA in spleen, corpus collosum, peripher al blood leukocytes, placenta, lung, aorta, and fetal tissues. Mouse S1P(5) mRNA is also expressed in spleen and brain. Finally, we found that one ena ntiomer of a sphingosine 1 -phosphate analogue wherein the 3-hydroxyl and 4 ,5-olefin are replaced by an amide functionality shows some selectivity as an agonist S1P(1) and S1P(3) vs S1P(2) and S1P(5).