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Vol. 60, Issue 5, 1064-1075, November 2001
Department of Pharmacology, Temple University School of Medicine,
Philadelphia, Pennsylvania
We demonstrated previously that D3.49(164) mutations resulted in
constitutive activation of the rat µ-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
[3H]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°C, and naloxone and
[D-Ala2,N-Me-Phe4,Gly5-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.
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