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, ...

The phospholipid growth factors sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are ligands for the related G protein-coupled receptors S1P 1 /EDG1 and LPA 1 /EDG2, respectively. We have developed a model of LPA 1 that predicts interactions between three polar residues and LPA. One of these, glutamine 125, which is conserved in the LPA receptor subfamily (LPA 1 /EDG2, LPA 2 /EDG4, and LPA 3 /EDG7), hydrogen bonds with the LPA hydroxyl group. Our previous S1P 1 study identified that the corresponding glutamate residue, conserved in all S1P receptors, ion pairs with the S1P ammonium. These two results predict that this residue might influence ligand recognition and specificity. Characterization of glutamate/glutamine interchange point mutants of S1P 1 and LPA 1 validated this prediction as the presence of glutamate was required for S1P recognition, whereas LPA recognition was possible with either glutamine or glutamate. The most likely explanation for this dual specificity behavior is a shift in the equilibrium between the acid and conjugate base forms of glutamic acid due to other amino acids surrounding that position in LPA 1 , producing a mixture of receptors including those having an anionic glutamate that recognize S1P and others with a neutral glutamic acid that recognize LPA. Thus, computational modeling of these receptors provided valid information necessary for understanding the molecular pharmacology of these receptors. Lysophosphatidic acid (LPA)1 and sphingosine-1-phosphate (S1P, see Fig. 1A) are members of the phospholipid growth factor family (for reviews, see Refs. 1-3). The responses elicited by phospholipid growth factors are pleiotropic and include the enhancement of cell survival, induction of cell proliferation, regulation of the actin-based cytoskeleton affecting cell shape, adherence, and chemotaxis, and the activation of Cl Ϫ and Ca 2ϩ ion conductances. LPA has been implicated in a number of disease and injury states, due to elevated levels of LPA in fluids surrounding the tissues involved, including corneal injury, lung disease, atherosclerosis, ovarian cancer, and wound healing. The eight receptors in the endothelial differentiation gene (EDG) family encode G protein-coupled receptors activated by the phospholipid growth factors LPA and S1P (4, 5). The EDG family is subdivided into two clusters based on ligand selectivity. S1P 1/3/2/4/5 receptors (formerly EDG1/3/5/6/8) are specifically activated by S1P (4), whereas LPA 1/2/3 receptors (formerly EDG2/4/7) are specifically activated by LPA (5). Members of the S1P receptor subfamily display 40 -50% sequence identity to each other and 30 -35% identity to the members of the LPA receptor subfamily (5, 6). These homologies and a distant relatedness to the cannabinoid receptors (7,8) suggest that the LPA-and S1P-specific subfamilies may have evolved from a common ancestral lipid receptor through the evolutionary development of distinct ligand binding pockets. If so, ligand selectivity should be determined by a limited number of cons...

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In this study, we have developed a two receptor model system to describe the R and R states of G-protein coupled receptors, specifically the alpha(1D) adrenergic receptor. The two models interact with agonist (epinephrine) and antagonist (BMY7378) differently. The active model has increased interactions with epinephrine. The inactive model has increased interactions with BMY7378. We also explored the protonation state of the ligands. When the most basic amine was protonated, we found increased hydrogen bonding and increased aromatic interactions. Protonated epinephrine hydrogen bonds with Asp176 and has aromatic residues Trp172, Trp235, Trp361, and Phe388 within 3 Angstroms. Protonated BMY7378 hydrogen bonds with Trp172 and Lys236 and has aromatic residues Trp172, Trp254, Phe364, Phe384, and Phe388 within 3 Angstroms. We conclude that the two model system is required to represent the two states of the receptor and that protonation of the ligand is also critical.

G protein-coupled receptors (GPCRs) comprise a large class of transmembrane proteins that play critical roles in both normal physiology and pathophysiology. These critical roles offer targets for therapeutic intervention, as exemplified by the substantial fraction of current pharmaceutical agents that target members of this family. Tremendous contributions to our understanding of GPCR structure and dynamics have come from both indirect and direct structural characterization techniques. Key features of GPCR conformations derived from both types of characterization techniques are reviewed.

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