Abstract
Phosphorylation of the μ-opioid receptor (MOR) is known as a key step in desensitization and internalization but the role in the development of long-term tolerance at the cellular level is not known. Viral expression of wild type (exWT) and mutant MORs, where all phosphorylation sites on the C-terminus (Total Phosphorylation Deficient (TPD)) were mutated to alanine, were examined in locus coeruleus neurons in a MOR knockout rat. Both receptors activated potassium conductance similar to endogenous receptors in wild type animals. The exWT receptors, like endogenous receptors, acutely desensitized, internalized and, after chronic morphine treatment, displayed signs of tolerance. However, TPD receptors did not desensitize or internalize with agonist treatment. In addition the TPD receptors did not develop cellular tolerance following chronic morphine treatment. Thus C-terminal phosphorylation is necessary for the expression of acute desensitization, trafficking and one sign of long-term tolerance to morphine at the cellular level.
Highlights
Considerable effort has been aimed at characterizing the mechanisms that underlie acute m-opioid receptor (MOR) dependent desensitization and cellular tolerance (Williams et al, 2013)
Microinjections of adeno-associated virus type two containing either the expression of wild type (exWT) or total phosphorylation deficient receptors (TPD) receptors were made bilaterally into the locus coeruleus (LC) and after 2–4 weeks the green fluorescence protein (GFP) tagged MORs were visualized with an Olympus macroview microscope (Figure 3A)
Phosphorylation of the C-terminal of the MOR has been shown to be a key step in the mechanism of acute desensitization measured with multiple downstream effectors
Summary
Considerable effort has been aimed at characterizing the mechanisms that underlie acute m-opioid receptor (MOR) dependent desensitization and cellular tolerance (Williams et al, 2013). Point mutations of serine (S) and threonine (T) residues in the STANT sequence resulted in a decrease in agonist induced arrestin recruitment and internalization, but did not eliminate the induction of acute desensitization (Lau et al, 2011; Birdsong et al, 2015). The TSST and STANT sequences are known to be agonist-dependent phosphorylation sites, there are four other serine and threonine sites on the C-terminus that are either phosphorylated constitutively or by agonist dependent kinases (Williams et al, 2013). It is not known if phosphorylation of these sites alters the regulation of MORs
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