FTY720 (Gilenya) Phosphate Selectivity of Sphingosine 1-Phosphate Receptor Subtype 1 (S1P1) G Protein-coupled Receptor Requires Motifs in Intracellular Loop 1 and Transmembrane Domain 2

FTY720 phosphate (FTY720P) is a high potency agonist for all the endothelial differentiation gene family sphingosine 1-phosphate (S1P) receptors except S1P receptor subtype 2 (S1P2). To map the distinguishing features of S1P2 ligand recognition, we applied a computational modeling-guided mutagenesis strategy that was based on the high degree of sequence homology between S1P1 and S1P2. S1P2 point mutants of the ligand-binding pocket were characterized. The head group-interacting residues Arg3.28, Glu3.29, and Lys7.34 were essential for activation. Mutation of residues Ala3.32, Leu3.36, Val5.41, Phe6.44, Trp6.48, Ser7.42, and Ser7.46, predicted to interact with the S1P hydrophobic tail, impaired activation by S1P. Replacing individual or multiple residues in the ligand-binding pocket of S1P2 with S1P1 sequence did not impart activation by FTY720P. Chimeric S1P1/S1P2 receptors were generated and characterized for activation by S1P or FTY720P. The S1P2 chimera with S1P1 sequence from the N terminus to transmembrane domain 2 (TM2) was activated by FTY720P, and the S1P2(IC1-TM2)S1P1 domain insertion chimera showed S1P1-like activation. Twelve residues in this domain, distributed in four motifs a–d, differ between S1P1 and S1P2. Insertion of 78RPMYY in motif b alone or simultaneous swapping of five other residues in motifs c and d from S1P1 into S1P2 introduced FTY720P responsiveness. Molecular dynamics calculations indicate that FTY720P binding selectivity is a function of the entropic contribution to the binding free energy rather than enthalpic contributions and that preferred agonists retain substantial flexibility when bound. After exposure to FTY720P, the S1P2(IC1-TM2)S1P1 receptor recycled to the plasma membrane, indicating that additional structural elements are required for the selective degradative trafficking of S1P1.