Abstract
Sphingosine 1-phosphate (S1P) influences heart rate, coronary artery caliber, endothelial integrity, and lymphocyte recirculation through five related high affinity G-protein-coupled receptors. Inhibition of lymphocyte recirculation by non-selective S1P receptor agonists produces clinical immunosuppression preventing transplant rejection but is associated with transient bradycardia. Understanding the contribution of individual receptors has been limited by the embryonic lethality of the S1P(1) knock-out and the unavailability of selective agonists or antagonists. A potent, S1P(1)-receptor selective agonist structurally unrelated to S1P was found to activate multiple signals triggered by S1P, including guanosine 5'-3-O-(thio)triphosphate binding, calcium flux, Akt and ERK1/2 phosphorylation, and stimulation of migration of S1P(1)- but not S1P(3)-expressing cells in vitro. The agonist also alters lymphocyte trafficking in vivo. Use of selective agonism together with deletant mice lacking S1P(3) receptor reveals that agonism of S1P(1) receptor alone is sufficient to control lymphocyte recirculation. Moreover, S1P(1) receptor agonist plasma levels are causally associated with induction and maintenance of lymphopenia. S1P(3), and not S1P(1), is directly implicated in sinus bradycardia. The sustained bradycardia induced by S1P receptor non-selective immunosuppressive agonists in wild-type mice is abolished in S1P(3)-/- mice, whereas S1P(1)-selective agonist does not produce bradycardia. Separation of receptor subtype usage for control of lymphocyte recirculation and heart rate may allow the identification of selective immunosuppressive S1P(1) receptor agonists with an enhanced therapeutic window. S1P(1)-selective agonists will be of broad utility in understanding cell functions in vitro, and vascular physiology in vivo, and the success of the chemical approach for S1P(1) suggests that selective tools for the resolution of function across this broad lipid receptor family are now possible.
Highlights
Sphingosine 1-phosphate (S1P) receptor function in the immune system has experimental advantages in approaching selective S1P receptor function at vascular interfaces
Published data on FTY720 phosphonate (respective IC50 values for human S1P1 (8.2 nM), S1P2 (Ͼ10,000 nM), S1P3 (151 nM), S1P4 (33 nM), and S1P5 (178 nM)) suggested that S1P1 is responsible for inhibition of lymphocyte egress [9], a fact that was subsequently strengthened by structure-activity correlations among a collection of semi-selective S1PR agonists [43, 44]
FTY720 binding implies that G-protein-coupled receptor privileged structures, structurally unrelated to S1P, could likely access the transmembrane site as agonists, with sequence differences between receptor subtypes making the discovery of selective agonists probable [30]
Summary
Membranes were prepared from CHO cells expressing human or murine S1P1, S1P2, S1P3, S1P4, and S1P5, for use in ligand and [35S]GTP␥S binding studies as described previously [9] and suspended in Buffer B with 15% glycerol and stored at Ϫ80 °C
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