This item is accessible only to the Washington University community.

Off-Campus WUSTL Users: Click the “Off-Campus Download” button below. You will be prompted to log in using your WUSTL Key.

Date Submitted

Fall 10-13-2014

Research Mentor and Department

Michael R. Bruchas

Restricted/Unrestricted

Dissertation/Thesis

Abstract

Traditional opioid receptors such as the μ and κ opioid receptors (MOR and

KOR) are well studied and are found to be common targets for pain and

stress therapies. The nociceptin opioid receptor (NOPR/ORL1) is a most

recently discovered opioid receptor whose complete function is still relatively

unknown, however is of great interest to researchers due to its analgesic

and antidepressant properties. NOPR activation due to ligand binding

triggers a variety of downstream effects, including G-protein signaling,

arrestin-mediated signaling, and receptor internalization. A variety of NOPR

ligands, both putative agonists and antagonists, were characterized by

further examination of these pathways. We performed ligand internalization

assays utilizing YFP-transfected receptors in order to visualize the

internalization with confocal microscopy. The ligands were also

characterized using protein immunoblots, evaluating the levels of c-JUN-N

terminal Kinase (JNK) and Extracellular signal-regulated Kinase (ERK)

phosphorylation. Variables such as concentration and treatment time

provided a comprehensive analysis of the various NOPR-selective ligands.

By quantifying the effects that different ligands have on MAPK signal

pathways and receptor internalization, we were able to form a clear

understanding of the signaling potential of various classes of NOPR ligands,

thus elucidating how different ligands can drive different outcomes through

the same receptor. Using dose and treatment time as varying experimental

factors, potential discoveries for ideal opioid analgesic therapies may be

revealed.