Identification of the Hydrophobic Ligand Binding Pocket of the S1P1 Receptor

Abstract

Sphingosine 1-phosphate (S1P), a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type, is an endogenous ligand of five G protein-coupled receptors (GPCRs) in the endothelial differentiation gene family. The lack of GPCR crystal structures sets serious limitations to rational drug design and in silico searches for subtype-selective ligands. Here we report on the experimental validation of a computational model of the ligand binding pocket of the S1P 1 GPCR surrounding the aliphatic portion of S1P. The extensive mutagenesis-based validation confirmed 18 residues lining the hydrophobic ligand binding pocket, which, combined with the previously validated three head groupinteracting residues, now complete the mapping of the S1P ligand recognition site. We identified six mutants (L3.43G/ L3.44G, L3.43E/L3.44E, L5.52A, F5.48G, V6.40L, and F6.44G) that maintained wild type [ 32 P]S1P binding with abolished ligand-dependent activation by S1P. These data suggest a role for these amino acids in the conformational transition of S1P 1 to its activated state. Three aromatic mutations (F5.48Y, F6.44G, and W6.48A) result in differential activation, by S1P or SEW2871, indicating that structural differences between the two agonists can partially compensate for differences in the amino acid side chain. The now validated ligand binding pocket provided us with a pharmacophore model, which was used for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics chemical library, leading to the identification of two novel nonlipid agonists of S1P 1 .Sphingosine 1-phosphate (S1P)3 (see Fig. 1) is a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type (1-3). S1P plays fundamental physiological roles in vascular stabilization (4), heart development (5), lymphocyte homing (6), and cancer angiogenesis (7). S1P elicits its biological effects through the activation of G protein-coupled receptors (GPCR) (8 -10) and through yet undefined intracellular targets (11-15). The endothelial differentiation gene (EDG) family of GPCR encodes eight highly homologous receptors. Five of these receptors, designated S1P 1 -S1P 5 , are specific for S1P, and the other three, LPA 1 -LPA 3 , are specific for the related lysophospholipid mediator lysophosphatidic acid (LPA) (16).FTY-720, an immunosuppressive prodrug presently in phase 3 clinical trials, has attracted a lot of interest due to its effective inhibition of kidney transplant rejection and attenuation of autoimmune diseases, including multiple sclerosis (6,17,18). In vivo, FTY-720 becomes phosphorylated by sphingosine kinase type 2, and FTY-720-P is a high affinity ligand of all EDG family S1P receptors with the exception of S1P 2 (19). In atrial myocytes, FTY-720-P, similarly to S1P (20, 21), activates an inwardly rectifying K ϩ conductance through the activation of the S1P 3 receptor, which in turn elicits unwanted bradyca...

Molecular recognition in the sphingosine 1-phosphate receptor family

“…S1P 1 prefers a linear binding mode and has a higher affinity to unsaturated ligands while S1P 4 prefers a bent binding mode and saturated ligands 28,29,42 . Docking results show the hydrophobic tails of all studied ligands are extended when docked into S1P 1 and S1P 5 models, but folded in the S1P 4 binding pocket.…”

FTY720 (Gilenya) Phosphate Selectivity of Sphingosine 1-Phosphate Receptor Subtype 1 (S1P1) G Protein-coupled Receptor Requires Motifs in Intracellular Loop 1 and Transmembrane Domain 2

“…Flow Cytometric Analysis-Expression of receptor constructs on the cell surface was measured by flow cytometry using indirect immunofluorescence staining with anti-FLAG M2 antibody as described previously (26).…”

“…We have applied computational modeling-guided mutagenesis studies for mapping the common and distinguishing features of ligand recognition by endothelial differentiation gene family lysophosphatidic acid and S1P G protein-coupled receptors (22)(23)(24)(25)(26)(27). Previously, we have developed and validated a computational model of the ligand-binding pocket of S1P 1 that was used to successfully screen the NCI Developmental Therapeutics Library for two non-lipid S1P 1 agonists (26). In the present study, we set out to identify the structural basis of the lack of activation of S1P 2 by FTY720P.…”

The lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) receptor gene families: cloning and comparative expression analysis in Xenopus laevis

“…The residues S114 and T187, but not Y265, of LPA4 protein (based on human sequence) which hydrogen bond with the polar head group of LPA, are conserved in Xenopus proteins (Li et al 2009). The residues R78, H160, R261, but not R276, of LPA5 (based on human sequence) which are involved in ligand recognition, are conserved in Xenopus proteins (Williams et al 2009), Several residues which have been implicated in the binding of S1P to its human receptor S1P1 are conserved in the other mammal orthologs (Parrill et al 2000;Fujiwara et al 2007). Seven of these 10 are also present in all Xenopus receptors, especially R124 and E125 (based on the human sequence) which are essential for S1P binding (Parrill et al 2000).…”