Identification of Leu276 of the S1P1 receptor and Phe263 of the S1P3 receptor in interaction with receptor specific agonists by molecular modeling, site-directed mutagenesis, and affinity studies

Sphingosine-1-phosphate (S1P) receptor agonists are novel immunosuppressive agents. The selectivity of S1P 1 against S1P 3 is strongly correlated with lymphocyte sequestration and minimum acute toxicity and bradycardia. This study describes molecular modeling, site-directed mutagenesis, and affinity studies exploring the molecular basis for selectivity between S1P 1 and S1P 3 receptors. Computational models of human S1P 1 and S1P 3 receptors bound with two nonselective agonists or two S1P 1-selective agonists were developed based on the X-ray crystal structure of bovine rhodopsin. The models predict that S1P(1) Leu276 and S1P(3) Phe263 contribute to the S1P(1)/S1P(3) selectivity of the two S1P(1)-selective agonists. These residues were subjected to site-directed mutagenesis. The wild-type and mutant S1P receptors were expressed in Chinese hamster ovary cells and examined for their abilities to bind to and be activated by agonists in vitro. The results indicate that the mutations have minimal effects on the activities of the two nonselective agonists, although they have dramatic effects on the S1P(1)-selective agonists. These studies provide a fundamental understanding of how these two receptor-selective agonists bind to the S1P(1) and S1P(3) receptors, which should aid development of more selective S1P(1) receptor agonists with immunosuppressive properties and improved safety profiles.