Identification of Edg1 Receptor Residues That Recognize Sphingosine 1-Phosphate

Abstract

Originating from its DNA sequence, a computational model of the Edg1 receptor has been developed that predicts critical interactions with its ligand, sphingosine 1-phosphate. The basic amino acids Arg 120 and Arg 292 ion pair with the phosphate, whereas the acidic Glu 121 residue ion pairs with the ammonium moiety of sphingosine 1-phosphate. The requirement of these interactions for specific ligand recognition has been confirmed through examination of site-directed mutants by radioligand binding, ligand-induced [35 S]GTP␥S binding, and receptor internalization assays. These ion-pairing interactions explain the ligand specificity of the Edg1 receptor and provide insight into ligand specificity differences within the Edg receptor family. This computational map of the ligand binding pocket provides information necessary for understanding the molecular pharmacology of this receptor, thus underlining the potential of the computational method in predicting ligand-receptor interactions.The G protein-coupled receptor (GPCR) 1 superfamily includes more than 2000 genes encoding receptors for bioactive molecules ranging from hormones through neurotransmitters (1). The physiological significance of these ligands makes GPCRs the target of many drugs and the subject of drug development studies. GPCRs are integral membrane proteins with physical properties that vastly increase the difficulty of standard methods of structure analysis. A precise understanding of their ligand-receptor interactions is essential for the rational design of ligands. Therefore, an atomic resolution map of the binding pocket including the locations of the interactions necessary for ligand binding would provide crucial drug development information. The endothelial differentiation gene (Edg) receptors are GPCRs that are activated by lysophospholipids (Fig. 1). Five members of the Edg family (Edg1, Edg3, Edg5, Edg6, and Edg8) show a preference for sphingosine 1-phosphate (SPP) (2-7). The remaining three Edg family members (Edg2, Edg4, and Edg7) are activated by lysophosphatidic acid (LPA) (8, 9). The receptor-mediated effects of SPP include stimulation of cell proliferation, prevention of apoptosis, regulation of cell shape, adhesion, motility, vascular differentiation (10 -12), and cancer cell invasiveness (11, 13). Therefore, receptors for SPP are important targets for the design of receptor-specific ligands both as potential therapeutic agents and to assist in the elucidation of the physiological function of the receptor.Membership of the Edg receptors in the GPCR family confers significant homology with other GPCRs in regard to sequence, topology, and function (14). These shared features make the Edg receptors good candidates for homology modeling. Homology modeling uses a known template protein structure to build an analogous structure for a protein sequence having unknown structure. Homology modeling thus assumes that homologous function and amino acid sequences confer three-dimensional structural similarity. GPCRs have extracellular amino termini, ...

Sphingosine 1-Phosphate (S1P) Receptor Subtypes S1P1 and S1P3, Respectively, Regulate Lymphocyte Recirculation and Heart Rate

“…Our published data (11) showed that total plasma concentrations at the ED 100 for lymphopenia for AFD-(R) were 20.6 nM, ϳ16-fold the EC 50 in vitro, whereas the same ratio calculated for SEW2871 from the plasma pharmacokinetics required ϳ300-fold the EC 50 to reach the ED 100 despite the fact that SEW2871 is only 65% bound in plasma, compared with AFD-(R), which is 96.8% plasma-bound (11). This could be explained in part by the 15-fold loss of potency of SEW2871 compared with AFD-(R), although it remains possible that the headgroup interactions contribute both potency and agonist efficiency (28). Quantitative evaluation of analogs of SEW2871 with headgroups attached may provide the answer.…”

“…High Throughput Screening Identifies S1P 1 -selective Agonists-Published binding studies on hS1P 1 with FTY720 and FTY-P (9), as well as mutagenesis and modeling with natural ligand S1P (28,29), suggested a two-site binding model. The hydrophobic-aromatic residues bind within receptor transmembrane domains and the ligand headgroups form salt bridges with glutamate and arginine side chains.…”

Prediction of structure and function of G protein-coupled receptors

“…Experimental data relevant to the function of GPCRs is available for ligand activation of GPCRs (7-15) and site-directed mutagenesis (16)(17)(18). This data has led to information about structural features in the ligand-binding regions of GPCRs (refs.…”

“…This is because these membrane-bound proteins are difficult to crystallize, and the atomic-level structure has been solved only for bovine rhodopsin (3, 4). Consequently, it is important to develop theoretical methods to predict the structure and function of GPCRs (5, 6).Experimental data relevant to the function of GPCRs is available for ligand activation of GPCRs (7-15) and site-directed mutagenesis (16)(17)(18). This data has led to information about structural features in the ligand-binding regions of GPCRs (refs.…”

Effects of Synthetic Sphingosine-1-Phosphate Analogs on Cytosolic Phospholipase A2α–Independent Release of Arachidonic Acid and Cell Toxicity in L929 Fibrosarcoma Cells: the Structure–Activity Relationship

“…phosphate group, a N-Boc group, and a phenyl group with a meta C 11 saturated carbon chain, could markedly release AA from L929 cells. D-erythro-O,O-Dimethyl-S1P is inactive upon binding to cells expressing S1P receptors such as S1P 1-3 receptors (32), and the protonated amino group of S1P is critical for the recognition of S1P by the S1P 1 receptor (33). Methylation of the C1 phosphate group and the C2 amino-group of S1P eliminate S1P's ability to interact with S1P receptors.…”