Inverse agonist up-regulates the constitutively active D3.49(164)Q mutant of the rat mu-opioid receptor by stabilizing the structure and blocking constitutive internalization and down-regulation.

We demonstrated previously that D3.49(164) mutations resulted in constitutive activation of the rat mu-opioid receptor and abolished receptor expression unless cells were pretreated with naloxone, an inverse agonist. In this study, we investigated the properties of the D3.49(164)Q mutant and the mechanisms underlying the effect of naloxone. Naloxone pretreatment up-regulated [(3)H]diprenorphine binding and protein expression of the D3.49(164)Q mutant in a time- and dose-dependent manner without affecting its mRNA level. After naloxone removal, binding and protein expression of the mutant declined with time with no effect on its mRNA level. Naloxone methiodide (a quaternary ammonium analog) caused a maximal up-regulation about 50% of the naloxone effect, indicating that naloxone acts extracellularly and intracellularly. Expression of the mutant was enhanced by inverse agonists, a neutral antagonist, and agonists, with inverse agonists being most effective. In membranes, the mutant was structurally less stable than the wild type upon incubation at 37 degrees C, and naloxone and [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin stabilized the mutant. Coexpression of the dominant-negative mutants GRK2-K220R, arrestin-2(319-418), dynamin I-K44A, rab5A-N133I or rab7-N125I partially prevented the decline in binding of the mutant after naloxone removal. Chloroquine or proteasome inhibitor I reduced the down-regulation of the mutant. These results indicate that the D3.49(164)Q mutant is constitutively internalized via G protein coupled-receptor kinase-, arrestin-2-, dynamin-, rab5-, and rab7-dependent pathways and probably trafficked through early and late endosomes into lysosomes and degraded by lysosomes and proteasomes. Naloxone up-regulates the D3.49(164)Q mutant by stabilizing the mutant protein and blocking its constitutive internalization and down-regulation. To the best of our knowledge, this represents the first comprehensive analysis of the mechanisms involved in up-regulation of constitutively active mutants by an inverse agonist.