A Select Set of Opioid Ligands Induce Up-Regulation by Promoting the Maturation and Stability of the Rat κ-Opioid Receptor in Human Embryonic Kidney 293 Cells

Abstract

Ligand-induced regulation of the rat -opioid receptor (rKOR) was investigated in human embryonic kidney 293 cells stably expressing the FLAG-tagged rKOR. Incubation of rKOR cells with naltrexone for 24 h increased the B max Ͼ3-fold, with no change in the affinity of [ 3 H]diprenorphine. Two immunoreactive receptor species were present in cell lysates: naltrexone treatment caused a Ͼ3-fold increase in the 52-kDa species while decreasing the level of the 42-kDa species. Dynorphin(1-13), U69,593]pyran-7-carboxylic acid methyl ester] treatment did not alter the level of immunoreactive rKOR protein, whereas etorphine, cyclazocine, naloxone, and naloxone methiodide increased the 52-kDa and decreased the 42-kDa rKOR bands. Receptor up-regulation was associated with an increase in the number of cell surface receptors and a 2-fold increase in the E max for guanosine 5Ј-O-(3-[ 35 S]thio)triphosphate binding. Glycosidase digestion indicated that the 52-and 42-kDa receptors contained complex and high-mannose N-glycans, respectively, Pulse-chase analysis and glycosidase digestion sensitivities suggested that the 42-kDa rKOR species was a precursor of the 52-kDa species. Naltrexone did not alter rKOR mRNA levels or translational efficiency, and rKOR up-regulation was not inhibited by cycloheximide. Brefeldin A caused accumulation of intracellular rKOR intermediates, and coincubation with naltrexone increased the levels of the brefeldin-induced species significantly. These results suggest that select opioid ligands upregulate rKOR by enhancing the rate of receptor folding and maturation and by protecting the receptor from degradation, resulting in an increase in the number of rKOR binding sites, immunoreactive protein, and functional receptors.Chronic administration of morphine or other opioid agonists for pain relief results in tolerance to the analgesic effects and physical dependence (McQuay, 1999). In contrast, chronic blockade of opioid receptors with antagonists such as naloxone and naltrexone does not result in physical dependence and was shown nearly 30 years ago to be associated with supersensitivity to the analgesic actions of morphine (Tang and Collins, 1978). The enhanced analgesic effects of morphine following infusion of naloxone were correlated with an increase in the number of opioid receptor binding sites (Lahti and Collins, 1978). Antagonist-mediated opioid receptor up-regulation has been shown to occur in vivo for -opioid receptors (MORs), ␦-opioid receptors, and -opioid receptors (KORs) (Morris et al., 1988;Lesscher et al., 2003), highlighting its biological relevance; however, the underlying mechanisms remain unclear. The steady-state level of opioid receptor mRNAs did not change in response to chronic in vivo antagonist treatment; therefore, post-transcriptional mechanisms are apparently involved (for review, see Unterwald and Howells, 2008).A large number of human diseases, for example cystic fibrosis, are due to genetic mutations that cause protein misfolding (Thomas et al

Purification and mass spectrometric analysis of the κ opioid receptor

“…The ultracentrifugal supernatant, containing approximately 300 mg of solubilized membrane protein, was incubated with WGA agarose beads to enrich the 52 kDa mature form of rKOR. We have shown previously that the 42 kDa does not bind efficiently to WGA (Wannemacher et al, 2007). Aliquots (0.03% v/v) of the WGA-agarose input, flow-through fraction and N-acetylglucosamine eluate were resolved using SDS/PAGE to assess the effectiveness of the lectin chromatography to purify the receptor.…”

“…Surprisingly, chronic agonist treatment leads to receptor down-regulation in the case of the human, but not the mouse or rat KOR (Zhang et al, 2002; Chen et al, 2006; Wannemacher et al, 2007). The carboxyl terminus of the receptor is believed to contribute towards the species variation (Zhang et al, 2002).…”

“…Down-regulation of the human κ-opioid receptor has also been shown to be partially attenuated by co-treatment with a proteasome inhibitor (Li et al, 2000), and polyubiquitination of the receptor is associated with down-regulation (Li et al, 2008). Additionally, we have provided evidence that the proteasome is also involved in basal turnover of the rat κ-opioid receptor (Wannemacher et al, 2007). …”

The Interaction Between the Mu Opioid Receptor and Filamin A

“…Though this may be the first instance of agonist-caused upregulation of MOPr in cell culture, this phenomenon has been observed with other GPCRs, including the kappa opioid receptor, where the kappa agonist cyclazozine and the ''universal'' opioid agonist etorphine can induce upregulation in HEK293 cell cultures [25]. Several laboratories have reported an increase in D2 (but not D1) dopamine receptors after chronic agonist exposure [26][27][28][29].…”

Chaperone-like effects of cell-permeant ligands on opioid receptors

“…(37) reported that a 24-h treatment with etorphine and cyclazocine significantly promoted maturation of rat KOPRs (rKOPRs), but treatment with U69,593 and salvinorin A did not, although all four agonists are cell-permeant and rKOPRs are not internalized and degraded following receptor activation. Thus, cell-permeant ligands showed differential abilities to act as pharmacological chaperones.…”