Investigating the molecular mechanisms through which FTY720‐P causes persistent S1P₁ receptor internalization

Abstract

BACKGROUND AND PURPOSEThe molecular mechanism underlying the clinical efficacy of FTY720-P is thought to involve persistent internalization and enhanced degradation of the S1P1 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 FTY720-P. release. CHO-S1P1/3R numbers were determined, following FTY720-P treatment using flow cytometry. EXPERIMENTAL APPROACH KEY RESULTSThe kinetic off-rate of [ 3 H]-FTY720-P from the S1P1R was sixfold slower than from the S1P3R, and comparable to [ 33 P]-S1P dissociation from S1P1/3Rs. S1P and FTY720-P stimulated [ 35 S]-GTPγS incorporation to similar degrees, but FTY720-P was over 30-fold less potent at S1P3Rs. FTY720-P stimulated a higher level of β-arrestin recruitment at S1P1Rs, 132% of the total recruited by S1P. In contrast, FTY720-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 FTY720-P, which is metabolically stable unlike S1P. CONCLUSIONS AND IMPLICATIONSFTY720-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 FTY720-P's ability to promote prolonged S1P1R internalization and may be critical factors in its efficacy in the clinic. AbbreviationsFTY720-P, 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol phosphate; MEM, minimal essential medium; NSB, non-specific binding; S1P, sphingosine 1-phosphate; SPA, scintillation proximity assay BJP British Journal of Pharmacology

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