Nontransformed cells depend on growth factors for their survival, proliferation and differentiation. In addition to polypeptide growth factors, an emerging group of naturally occurring group of lipid mediators, named phospholipid growth factors (PLGF) have been discovered. Within this group, lysophosphatidic acid (1-acyl-2-lyso-sn- glycero-3-phosphate, LPA), nature's simplest phospholipid, was found to elicit growth factor-like effects in almost every cell type spanning the phylogenetic tree, from Dictyostelium to humans. A structurally unrelated sphingolipid, sphingosine-l-phosphate also possesses PLGF- like effects. The immediate objective of this work is to understand how receptor structure defines ligand selectivity in the endothelial differentiation gene (EDG) PLGF receptor family. The EDG gene family, is a gene family which encodes PLGF receptors and currently consists of eight G protein-coupled receptors recognizing either lysophosphatidic acid or sphingosine-1-phosphate. Here we propose a mutagenesis based approach to understanding receptor ligand interactions in the EDG family which is guided by computational modeling, ligand binding, and receptor function studies. These experimental studies will refine and validate high-resolution molecular models of the individual EDG receptors, which can then be used to achieve our long-term goal aimed at developing therapeutically applicable PLGF analogs. This is an interdisciplinary proposal, which builds on the expertise of the PI in PLGF receptors and PLGF pharmacology and brings it together with the expertise of Dr. Abby Parrill in computational chemistry and of Dr. Duane Miller in synthetic lipid chemistry. Three specific aims will be pursued: 1. Develop and validate a refined computational model for the interaction of SPP with the EDG1 receptor. 2. Identify residues conferring specificity for sphingolipids in the EDG3/5/6/8 receptor subfamily. 3. Identify residues involved in glycerolipid recognition in the EDG2/4/7 receptor subfamily.