OR24-01 Mutational Study of the GPR119 Receptor Binding Site

Abstract

The GPR119 receptor, a class A G-protein coupled receptor located in the pancreatic β cells, induces insulin production when activated. Due to its specific activity, the pharmaceutical industry has identified GPR119 as a target for the treatment for type 2 diabetes. The lack of a GRP119 crystal structure has hindered the study of the receptor so our laboratory developed GPR119 active and inactive homology models. Docking studies with the inactive receptor model indicated that two leucine residues facing the binding pocket, L5.43(169) and L6.52(242), may be involved in ligand activation. Additionally, a serine at the extracellular end of the pocket, S1.32(4), may help orient of the ligand in the binding pocket via hydrogen bonding. To gain further insight into the role of these residues and the receptor activation mechanism, molecular dynamics (MD) simulations and in vitro cAMP assays of the wild type and mutant receptors were employed. The software NAMD employing the CHARMM force field was used to carry out MD simulations of the active receptor model bound with the agonist AR231453 embedded in a hydrated lipid bilayer. Preliminary results indicate that L6.52(242), located on transmembrane helix (TMH) 6, does not face directly into the binding site and does not interact with the ligand, while L5.43(169), located on TMH5, does face into the binding site, potentially interacting directly with the ligand. Also, S1.32(4), because of its extracellular location, is solvated instead of interacting with the ligand. The in vitro studies overall support the MD simulations. The mutations L6.52(242)M and L6.52(242)A appear to have minimal to no effect on agonist-induced cAMP production, compared to the wild type. In contrast, the L5.43(169)M and L5.43(169)A mutations decrease the potency of activation by AR231453, indicating that L5.43(169) changes the shape of the binding pocket, affecting ligand binding and activation. Finally, the cAMP assays show that the S1.32(4)A mutant also shows decreased activity compared to the wild type, implying that the ligand may be losing a hydrogen bonding interaction when S1.32(4) is mutated to alanine.