Print version
Click to downloadSearch Pub Med
Investigating the molecular mechanisms through which FTY720-P causes persistent S1P1 receptor internalisation FTY720 (fingolimod) is a novel therapy for the treatment of multiple sclerosis. FTY720 is phosphorylated in-vivo to form the potent S1P1, S1P3-5 receptor agonist FTY720-P. Previous studies have shown that FTY720-P is able to cause persistent internalisation of the S1P1, but not S1P3 receptor. The aim of this study was to investigate whether differences in β-arrestin recruitment and receptor binding kinetics could play a role in the persistent internalization of S1P1 receptor by FTY720-P. Ligand efficacy was assessed in a GTPγS binding assay (Sykes et al., 2009) whilst the PathHunter™ β-Arrestin assay (Riddy et al., 2010) was used to determine CHO-S1P1 and S1P3 cellï€ β-arrestin recruitment. [3H]-FTY720-P and [33P]-S1P were used to label CHO-S1P1/3 receptors and dissociation was initiated with an excess of unlabelled S1P or FTY720-P and GTP (1mM). Binding was carried out in HBSS containing 0.5% BSA and 0.1mM sodium orthovanadate at room temperature. Additionally, the metabolic stability of S1P and FTY720-P was assessed (Bradley et al., 2011) along with CHO-S1P1 and S1P3 cell surface receptor numbers (Mullershausen et al., 2009). In saturation binding assays [3H]-FTY720-P was shown to have a Kd of 0.289 ± 0.079 and 0.432 ± 0.101 nM at S1P1 and S1P3 receptors respectively, whilst [33P]-S1P itself had a higher affinity for S1P1 and S1P3 receptors, 0.197 ± 0.022 and 0.045 ± 0.006nM respectively. Kinetically the off-rate of [3H]-FTY720-P from the uncoupled form of the S1P1 receptor was significantly slower than from S1P3 receptors (t½ values of 18.97 and 3.07 min, respectively). In contrast S1P dissociation from both S1P1 and S1P3 receptors was comparable to FTY720-P dissociation from S1P1 receptors (23.88 & 13.97 min respectively). S1P and FTY720-P stimulated [35S]-GTPγS incorporation to similar degrees, but despite displaying similar affinities at these two receptors, FTY720-P was less potent at S1P3 receptors, suggesting that it is a lower efficacy agonist at this receptor (pEC50s 9.32 verses 8.48). Both S1P and FTY720-P recruited β-arrestin, however FTY720-P stimulated a higher maximal level of β-arrestin recruitment than S1P at S1P1 receptors, 132 % of the total β-arrestin recruited by S1P. In contrast, FTY720-P was a low partial agonist at S1P3, stimulating just 29 % of the total β-arrestin recruited by S1P. Internalisation experiments confirmed that cell surface expression of the S1P1 receptor was lower after a pulse exposure to FTY720-P than with S1P. Finally, S1P was degraded by membranes derived from CHO cells whereas FTY720-P was metabolically stable. We propose a three-step mechanism to explain the different receptor internalisation characteristics of FTY720-P and S1P on S1P1 and S1P3 receptors. FTY720-P activation of S1P1 receptors results in rapid receptor internalization, possibly as a consequence of an efficient recruitment of β-arrestin. The slow receptor dissociation rate means that FTY720-P remains bound to the S1P1 receptor as it is internalised, becoming trapped in the intracellular vesicles. Although the dissociation rate of S1P suggests it would also be internalised with the receptor, its metabolic instability means it is rapidly degraded, allowing the receptors to be recycled to the cell surface. FTY720-P does not cause the same degree of S1P3 receptor internalisation as it is less efficient at recruiting β-arrestin and also rapidly dissociates from the receptor so less becomes trapped in the internalised vesicles, allowing the receptor to recycle back to the cell surface3
Bradley ME et al.,(2011) EJP 672, 56-61 Mullershausen F et al., (2009) Nature Chem Biol 5(6) 428-434 Riddy D, et al (2010) http://www.pA2online.org/abstracts/Vol8Issue1abst099P Sykes DA et al (2009). Mol. Pharm. 76(3):543-51.
|
|