title: Investigating the molecular mechanisms through which FTY720-P causes persistent S1P1 receptor internalization.
abstract: OBJECTIVE
  The 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.
  
  
  METHODS
  [(3) H]-FTY720-P and [(33) P]-S1P were used to label CHO-S1P1/3Rs for binding studies. Ligand efficacy was assessed through [(35) S]-GTPγS binding and β-arrestin recruitment. Metabolic stability was evaluated using a bioassay measuring intracellular Ca(2+) release. CHO-S1P1/3R numbers were determined, following FTY720-P treatment using flow cytometry.
  
  
  RESULTS
  The 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
  FTY720-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.
authors.affiliation: Novartis Institutes for Biomedical Research, Horsham, West Sussex, UK.
authors.forename: David A
authors.initials: DA
authors.lastname: Sykes
authors.forename: Darren M
authors.initials: DM
authors.lastname: Riddy
authors.forename: Craig
authors.initials: C
authors.lastname: Stamp
authors.forename: Michelle E
authors.initials: ME
authors.lastname: Bradley
authors.forename: Neil
authors.initials: N
authors.lastname: McGuiness
authors.forename: Afrah
authors.initials: A
authors.lastname: Sattikar
authors.forename: Danilo
authors.initials: D
authors.lastname: Guerini
authors.forename: Ines
authors.initials: I
authors.lastname: Rodrigues
authors.forename: Albrecht
authors.initials: A
authors.lastname: Glaenzel
authors.forename: Mark R
authors.initials: MR
authors.lastname: Dowling
authors.forename: Florian
authors.initials: F
authors.lastname: Mullershausen
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Charlton
mesh_terms: D000818:Animals; D019390:Arrestins / Q000378:metabolism; D016466:CHO Cells; D003412:Cricetulus; D006801:Humans; D007700:Kinetics; D008246:Lysophospholipids / Q000494:pharmacology*; D010755:Organophosphates / Q000494:pharmacology*; D049349:Receptors, Lysosphingolipid / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; C525008:FTY 720P; D008246:Lysophospholipids; D010755:Organophosphates; D049349:Receptors, Lysosphingolipid; D000071557:beta-Arrestins; C060506:sphingosine 1-phosphate; D013110:Sphingosine