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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.