Family A G protein-coupled receptor ligand interactions: the role of the extracellular region

Abstract

Increasing interest in the role of the extracellular loops (ECL) in family A G protein-coupled receptor (GPCR) function stems from (i) the discovery that many allosteric modulators of alpha branch receptors interact with the ECL, (ii) predictions that this region forms an initial interaction site for orthosteric ligands, (iii) ECL mutations alter receptor signalling and (iv) the ECL interacts with peptide ligands from the gamma branch receptors. This thesis investigated the role of the ECL and surrounding regions in allosteric and orthosteric ligand interactions across family A GPCRs using the alpha branch beta2 adrenoceptor (b2AR) and the gamma branch complement 5a receptor (C5aR). THRX100361 and tacrine displayed positive modulation of 3H-dihydroalprenolol (DHA) dissociation from the b2AR but negative modulation of b2AR activation. Using docking and mutagenesis, an allosteric site comprised of residues from ECL2 (F193) and the top of helices 6 (H296) and 7 (K305 and Y308) was identified. Mutation of these residues to alanine reduced the modulatory actions of THRX100361 and tacrine. These mutations also increased the dissociation rate of 3H-DHA. This supports the existence of a metastable binding site on the b2AR as predicted in computational studies and that this region is important in orthosteric and allosteric interactions. PMX53 is potent inhibitor of the C5aR and has previously been described as a non-competitive antagonist in myeloperoxidase and calcium release assays. Surprisingly, in this study, PMX53 behaved as a competitive antagonist of C5a-mediated Gi activation. This discrepancy suggests that PMX53 has a slow off rate, resulting in hemi-equilibrium conditions in signalling assays with short incubation times and is in fact a competitive antagonist. Supporting this, docking of PMX53 into a homology model suggested interactions with ECL1-2 and helices 2,3,5,6-7 including residues involved in C5a binding. The affinity of PMX53 is 400 fold lower at the mouse compared to human and rat C5aR. Docking and sequence alignment suggested that 2 residues in ECL2 may contribute to this difference. The human to mouse mutation L187V has no effect, while D191N increased PMX53 potency, indicating that ECL2 may play a role in PMX53 binding but does account for the species variation in affinity. Further studies are required to fully understand PMX53 molecular mechanism of action. The data in this thesis suggest an important role for the ECL2 in family A GPCRs.