Abstract
Sphingosine-1-phosphate (S1P) receptor agonists are novel immunosuppressive agents. The selectivity of S1P1 against S1P3 is strongly correlated with lymphocyte sequestration and minimum acute toxicity and bradycardia. This study describes molecular modeling, site-directed mutagenesis, and affinity studies exploring the molecular basis for selectivity between S1P1 and S1P3 receptors. Computational models of human S1P1 and S1P3 receptors bound with two nonselective agonists or two S1P1-selective agonists were developed based on the X-ray crystal structure of bovine rhodopsin. The models predict that S1P1 Leu276 and S1P3 Phe263 contribute to the S1P1/S1P3 selectivity of the two S1P1-selective agonists. These residues were subjected to site-directed mutagenesis. The wild-type and mutant S1P receptors were expressed in Chinese hamster ovary cells and examined for their abilities to bind to and be activated by agonists in vitro. The results indicate that the mutations have minimal effects on the activities of the two nonselective agonists, although they have dramatic effects on the S1P1-selective agonists. These studies provide a fundamental understanding of how these two receptor-selective agonists bind to the S1P1 and S1P3 receptors, which should aid development of more selective S1P1 receptor agonists with immunosuppressive properties and improved safety profiles.
Footnotes
-
↵2 After the manuscript for this article was submitted, we noted the recent release of X-ray crystal structures of activated forms of rhodopsin, including bathorhodopsin (PDB entry 2G87) (Nakamichi and Okada, 2006a), lumirhodopsin (PDB entry 2HPY) (Nakamichi and Okada, 2006b), and a photoactivated deprotonated intermediate of rhodopsin (PDB entry 2I37) (Salom et al., 2006). Given the high structural similarities between bovine rhodopsin in the dark state and the activated forms, the use of the dark state rhodopsin (PDB entry 1F88) as a modeling template would seem to be a reasonable starting point for homology modeling of family A GPCRs bound to agonists and antagonists.
-
S.A.P. and Q.D. contributed equally to this work.
-
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
-
doi:10.1124/mol.106.029223.
-
ABBREVIATIONS: S1P, sphingosine-1-phosphate; LPA, lysophosphatidic acid; GPCR, G protein-coupled receptor; Edg, endothelial differentiation gene; TM, transmembrane helix; FTY720, 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol; FTY720-P, 2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butyl dihydrogen phosphate; compound A, 1-(3-methyl-4-{[4-phenyl-5-(trifluoromethyl)-2 thienyl]methoxy}benzyl)azetidine-3-carboxylic acid; compound B, 1-{4-[5-(4-isobutylphenyl)-1,2,4-oxadiazol-3-yl]benzyl}azetidine-3-carboxylic acid; CHO, Chinese hamster ovary; MMFF, Merck molecular force field; PCR, polymerase chain reaction; HT, hypoxanthine/thymidine; PDB, Protein Data Bank; TM, transmembrane; PBS, phosphate-buffered saline; GTPγS, guanosine 5′-O-(3-thio)triphosphate; FACS, fluorescence-activated cell sorting.
-
↵1 Current affiliation: Inexel Medical Strategy and Communication, Lawrenceville, New Jersey.
- Received July 26, 2006.
- Accepted December 4, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|