G-protein-coupled receptors (GPCRs) are major pharmaceutical drug targets due to their crucial role in cell signalling. The neurohypophysial peptide hormones [Arg⁸] vasopressin (AVP) and oxytocin (OT) signal through a subfamily of GPCRs, comprising the AVP receptors (V₁a R, V₁bR and V₂R) and the OT receptor (OTR). The aim of this work was to understand the molecular basis of receptor activation by defining exact contacts between agonist and receptor. Molecular modelling suggested that two conserved negatively-charged residues may be important for agonist binding and receptor activation. Interactions between agonists and the human AVP/OT receptors were probed using a combination of site-directed mutagenesis of the receptors and ligands incorporating modifications at specific points. The wild-type (WT) and mutant receptors were expressed in HEK 293T cells and pharmacologically characterised with respect to ligand binding, receptor activation, cell surface expression and ligand-induced internalisation when stimulated by endogenous, or modified, ligands. Mutual exchange of functional groups revealed direct interactions between AVP/OT/[Arg⁸] vasotocin (AVT; a chimera of AVP and OT) and the human AVP/OT receptors required for receptor activation. These studies advance current understanding of the molecular basis of receptor activation and have the potential to facilitate future rational drug design.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:720736 |
| Date | January 2017 |
| Creators | La-Borde, Penelope Jane |
| Publisher | University of Birmingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.bham.ac.uk//id/eprint/7639/ |
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