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FTY720‐PHOSPHATE (GILENYA) SELECTIVITY OF S1P1 GPCR REQUIRES MOTIFS IN INTRACELLULAR LOOP 1 AND TRANSMEMBRANE DOMAIN 2

FTY720 phosphate (FTY720P) is a high potency agonist for all the EDG‐family S1P receptors except 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 residues R3.28, E3.29, and K7.34 were essential for activation. Mutation of residues A3.32, L3.36, V5.41, F6.44, W6.48, S7.42, and S7.46, 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. The S1P2 chimera with S1P1 sequence from the N‐terminus to TM2 was activated by FTY720P and the S1P2(IC1‐TM2)S1P1 domain‐insertion chimera showed S1P1‐like activation. Twelve residues in this domain differ between S1P1 and S1P2, distributed in four motifs a–d. 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.