Agonist-dependent effects of mutations in the sphingosine-1-phosphate type 1 receptor

The sphingosine-1-phosphate type 1 (S1P 1) receptor is a new target in the treatment of auto-immune diseases as evidenced by the recent approval of FTY720 (Fingolimod). The ligand-binding pocket of the S1P 1 receptor has been generally characterised but detailed insight into ligand-specific differences is still lacking. The aim of the current study is to determine differences in ligand-induced S1P 1 receptor activation using an in silico guided site-directed mutagenesis strategy. S1P 1 mutant receptors (modifications of residues Y98 2.57, R120 3.28, F125 3.33) were probed with a chemically diverse set of S1P 1 agonists (S1P, dihydro-S1P (dhS1P), R-, S- and racemic FTY720-P, VPC24191, SEW2871). Mutation of the R 3.28 residue generally results in a reduction of the potency of all ligands although the synthetic ligands including FTY720-P are less sensitive to these mutations. The Y 2.57F mutation does not affect the potency of any of the ligands tested, but for all ligands except FTY720-P a significant decrease in potency is observed at the Y 2.57A mutant. The F 3.33A mutation significantly decreased the potency of FTY720-P and is detrimental for SEW2871 and VPC24191. The non-aromatic endogenous ligands S1P and dhS1P are less affected by this mutation. Our in silico guided mutagenesis studies identified new molecular determinants of ligand-induced S1P 1 receptor activation: 1) the flexibility of the polar head of the agonist to maintain a tight H-bond network with R 3.28 and 2) the ability of the agonist to make aromatic π-stacking interactions with F 3.33. Interestingly, FTY720-P has both chemical properties and is the only ligand that can efficiently activate the Y 2.57A mutant. ► The effect of the S1P 1 mutations studied are ligand-dependent. ► The sensitivity of ligands towards a mutation depends on the flexibility and aromaticity of a ligand. ► FTY720-P has both properties and is the only ligand that can efficiently activate the Y 2.57A mutant.