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]GTPgammaS 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.
Highlights
The G protein-coupled receptor (GPCR)1 superfamily includes more than 2000 genes encoding receptors for bioactive molecules ranging from hormones through neurotransmitters [1]
Three-dimensional Structure of Edg1 in Complex with sphingosine 1-phosphate (SPP)—A preliminary homology model of Edg1 was developed from a model of bovine rhodopsin [16] that is consistent with the 9-Å resolution structural data on the rhodopsin molecule
The third involves an anionic amino acid, Glu121, positioned within 2.5 Å of the positively charged ammonium group of the sphingosine backbone. These ion-pairing interactions can provide an explanation of why the Edg1 receptor shows a strong preference for SPP over lysophosphatidic acid (LPA), a glycerophospholipid that lacks an ammonium substituent
Summary
Materials—SPP and LPA were from Avanti Polar Lipids (Alabaster, AL). Anti-flag M2 antibody and horseradish peroxidase- or fluorescein isothiocyanate-labeled anti-mouse were purchased from Sigma. 25 different SPP-Edg complexes based on computer-generated random starting positions were generated with full ligand flexibility These geometries were evaluated using the criteria from our previous modeling studies using the incorrect Edg sequence Geometry optimization of the best complex obtained with the correct Edg-1 sequence (accession number AFF43420) was performed to a 0.01 root mean square gradient after manual optimization of ion-pairing interactions. 5 g of membrane protein from Edg receptor expressing RH7777 cells was incubated in 1.0 ml of binding buffer (50 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM MgCl2, 10 M GDP, 2 mM dithiothreitol, 0.1 nM [35S]GTP␥S (1191 Ci/mmol; Amersham Pharmacia Biotech)). RH7777 cells transfected with empty vector or plasmids containing Edg construct inserts were cultured on coverslips in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. Immunofluorescence staining was viewed by laser-scanning confocal fluorescence microscopy
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have