| The G protein-coupled receptor (GPCR) super family is the largest known class of molecular targets with proven therapeutic value. Among 800 members are encoded by the human genome, bovine rhodopsin is the only GPCR with a known structure, demonstrating that three-dimensional structures for GPCR are very difficult to obtain. Knowledge about GPCR structures mainly comes from homology models, mutagenesis data, and low resolution structural studies. Sphingosine 1-phosphate (S1P) receptors belong to the GPCR family. They are particularly interesting due to their critical roles in angiogenesis, apoptosis, cell differentiation, motility and survival, as well as lymphocytes migration and trafficking, and potential use as targets to treat diseases associated with cardiovascular system, immune disorder and cancer.; Our research focused on structural characterization of S1P receptors. We have developed and published models of mouse S1P4, human S1P 4 and S1P5 receptors based on our experimentally validated S1P1 model. The 3D-models were refined using energy minimization and molecular dynamics. The final mouse S1P4 model was validated experimentally and computationally by site-directed mutagenesis and docking studies. Mutations of R3.28, K5.38, E3.29 and W4.64 abolish specific S1P binding and S1P-induced receptor activation.; Although structures of the transmembrane domains (TM) are well defined in the homology models, the loop conformations remain uncertain due to their high flexibility and low sequence identity. Thus, we developed a methodology for structurally characterizing loops, using the S1P4 first extracellular loop (E1) as a model system. This methodology uses computationally designed peptides in combination with circular dichroism and NMR. The characterized peptides contain mimetics of TM 2 at the N-terminus, E1 and mimetics of TM3 at the C-terminus including residues R3.28 and E3.29, which are required for ligand binding and receptor activation. Loop mimetic peptides were designed using several different strategies to promote structures relevant to the intact receptor. The disulfide crosslinked coiled-coil design adopted secondary structures indicated by chemical shift index (CSI) and heteronuclear NOE experiments most relevant to the intact protein. Its NMR structure in 20% trifluoroethanol has been solved. |
