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 constituti ve activation of the rat mu -opioid receptor and abolished receptor express ion 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 me chanisms underlying the effect of naloxone. Naloxone pretreatment upregulat ed [H-3]diprenorphine binding and protein expression of the D3.49(164)Q mut ant in a time- and dose-dependent manner without affecting its mRNA level. After naloxone removal, binding and protein expression of the mutant declin ed with time with no effect on its mRNA level. Naloxone methiodide (a quate rnary ammonium analog) caused a maximal up-regulation about 50% of the nalo xone effect, indicating that naloxone acts extracellularly and intracellula rly. Expression of the mutant was enhanced by inverse agonists, a neutral a ntagonist, and agonists, with inverse agonists being most effective. In mem branes, the mutant was structurally less stable than the wild type upon inc ubation at 37 degreesC, and naloxone and [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-e nkephalin stabilized the mutant. Coexpression of the dominant-negative muta nts GRK2-K220R, arrestin-2(319-418), dynamin I-K44A, rab5A-N133I or rab7-N1 25I 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 const itutively internalized via G protein coupled-receptor kinase-, arrestin-2-, dynamin-, rab5-, and rab7-dependent pathways and probably trafficked throu gh early and late endosomes into lysosomes and degraded by lysosomes and pr oteasomes. Naloxone up-regulates the D3.49(164)Q mutant by stabilizing the mutant protein and blocking its constitutive internalization and down-regul ation. To the best of our knowledge, this represents the first comprehensiv e analysis of the mechanisms involved in up-regulation of constitutively ac tive mutants by an inverse agonist.