Bohn, Laura

Opioid agonists are potent pain relievers that have serious side effects including respiratory suppression, dependence, and addiction. Biased agonism refers to a ligand dependent selectivity for specific down stream signaling pathways, which can be used as a model of receptor function in effort to develop safer analgesics. By changing the ligand, activated G-protein-coupled receptors can be manipulated to activate desired intracellular pathways, which results in positive clinical effects, e.g. analgesia, without activation of pathways that result in negative clinical effects, e.g. respiratory suppression and dependency. This thesis project will explore the in vitro and in vivo results of bias at the 𝜇-opioid receptor (𝜇OR), specifically investigating G-protein-preferential activation with reduced βarrestin2 recruitment, a regulatory protein responsible for many negative effects.

Opioids are considered the most efficacious drugs for management of moderate to severe pain, yet their use clinically is often restricted due to the onset of adverse side-effects. Drugs in this class produce most of their physiological effects (analgesia, nausea, vomiting) through activation of the μ-opioid receptor; however, an increasing number of studies have demonstrated that opioids can activate distinct downstream responses, a phenomenon termed functional selectivity. This project attempts to determine the amino acid residues involved in the activation of the μ-opioid receptor and the role they play in the binding of ligands to the receptor. The highly conserved DRY motif (aspartic acid-arginine-tyrosine) found commonly in GPCRs was mutated via site directed mutagenesis, aspartic acid-164 to alanine (A), glutamic acid (E), and arginine (R) and G-protein coupling and competition binding assays were performed to test whether ligand affinity and activity differed in the mutant receptors compared to the wild-type.