Investigating the molecular mechanisms through which FTY 720‐ P causes persistent S1P 1 receptor internalization

Background and Purpose The molecular mechanism underlying the clinical efficacy of FTY 720‐ P is thought to involve persistent internalization and enhanced degradation of the S1P 1 receptor subtype ( S1P1R ). We have investigated whether receptor binding kinetics and β‐arrestin recruitment could play a role in the persistent internalization of the S1P1R by FTY 720‐ P . Experimental Approach [ 3 H ]‐ FTY 720‐ P and [ 33 P ]‐ S1P were used to label CHO ‐ S1P1 / 3Rs for binding studies. Ligand efficacy was assessed through [ 35 S ]‐ GTP γ S binding and β‐arrestin recruitment. Metabolic stability was evaluated using a bioassay measuring intracellular Ca 2+ release. CHO ‐ S1P1 / 3R numbers were determined, following FTY 720‐ P treatment using flow cytometry. Key Results The kinetic off‐rate of [ 3 H ]‐ FTY 720‐ P from the S1P1R was sixfold slower than from the S1P3R , and comparable to [ 33 P ]‐ S1P dissociation from S1P1 / 3Rs . S1P and FTY 720‐ P stimulated [ 35 S ]‐ GTP γ S incorporation to similar degrees, but FTY 720‐ P was over 30‐fold less potent at S1P3Rs . FTY 720‐ P stimulated a higher level of β‐arrestin recruitment at S1P1R s, 132% of the total recruited by S1P . In contrast, FTY 720‐ P was a weak partial agonist at S1P3R , stimulating just 29% of the total β‐arrestin recruited by S1P . Internalization experiments confirmed that cell surface expression of the S1P1R but not the S1P3R was reduced following a pulse exposure to FTY 720‐ P , which is metabolically stable unlike S1P . Conclusions and Implications FTY 720‐ P and S1P activation of the S1P1R results in receptor internalization as a consequence of an efficient recruitment of β‐arrestin. The combination of slow off‐rate, efficacious β‐arrestin recruitment and metabolic stability all contribute to FTY 720‐ P 's ability to promote prolonged S1P1R internalization and may be critical factors in its efficacy in the clinic.