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Molecular mechanisms underlying antagonist-mediated opioid receptor up-regulation

by
Kenneth M. Wannemacher
Biochemistry and Molecular Biology

B.S. 2003 University of Delaware

Thesis Advisor: Richard Howells, Ph.D.
Professor
Biochemistry and Molecular Biology


MSB E609b

Friday, September 19, 2008
12:000 p.m.


Abstract

Chronic treatment with opioid receptor antagonists have been shown to up-regulate opioid receptor binding, as well as lead to supersensitivity to subsequent agonist treatment. Although these initial observations were made thirty years ago, the molecular mechanism remains unclear. Through the use of clonal human embryonic kidney 293 (HEK 293) cell lines stably expressing kappa or delta opioid receptor with an N-terminal FLAG epitope, we have identified mechanisms by which opioid receptor antagonists, such as naltrexone, are able to up-regulate functional opioid receptors. First, we have found that treatment of HEK 293 cells expressing the rat kappa opioid receptor (rKOR) with naltrexone leads to time-dependent increases in both ligand-receptor binding, as well as rKOR protein. Further studies have shown that a subset of both kappa antagonists and agonists are able to permeate the cell and bind to immature receptors in the ER. This provides protection from proteasome-mediate degradation and enhances the receptor folding rate and maturation through the secretory pathway, thus leading to an increase in functional rKOR at the cell surface.
Studies involving the murine delta opioid receptor (mDOR) have revealed an additional mechanism of action of antagonists. Treatment of mDOR-expressing HEK 293 cells with naltrexone causes a time-dependent increase in 3H-diprenorphine maximum binding without changing mDOR protein levels. Metabolic labeling studies have suggested that untreated mDOR is very efficiently processed through the secretory pathway with minimal degradation. Generation of maturation-deficient mutant receptors indicated that naltrexone could act as a pharmacological chaperone if receptor maturation was compromised. In wild type mDOR, the chaperone function of naltrexone is not required for receptor maturation, yet naltrexone is still able to increase the number of functional receptors without increasing the amount of mDOR protein. We hypothesize that naltrexone is able to bind to low affinity receptors present at the cell surface and induce a conformational change that promotes high affinity ligand binding. Taken together, our studies have revealed mechanisms by which antagonists can up-regulate functional opioid receptors. These studies provide useful information towards the further development of opioid receptor antagonists for therapeutic purposes.


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