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. 2003 Dec 1:3:14.
doi: 10.1186/1471-2210-3-14.

Enhanced spontaneous activity of the mu opioid receptor by cysteine mutations: characterization of a tool for inverse agonist screening

Karl Brillet  1 , Brigitte L Kieffer, Dominique Massotte
Affiliations

Enhanced spontaneous activity of the mu opioid receptor by cysteine mutations: characterization of a tool for inverse agonist screening

Karl Brillet et al. BMC Pharmacol. .

Abstract

Background: The concept of spontaneous- or constitutive-activity has become widely accepted and verified for numerous G protein-coupled receptors and this ligand-independent activity is also acknowledged to play a role in some pathologies. Constitutive activity has been reported for the mu opioid receptor. In some cases the increase in receptor basal activity was induced by chronic morphine administration suggesting that constitutive activity may contribute to the development of drug tolerance and dependence. Constitutively active mutants represent excellent tools for gathering information about the mechanisms of receptor activation and the possible physiological relevance of spontaneous receptor activity. The high basal level of activity of these mutants also allows for easier identification of inverse agonists, defined as ligands able to suppress spontaneous receptor activity, and leads to a better comprehension of their modulatory effects as well as possible in vivo use.

Results: Cysteines 348 and 353 of the human mu opioid receptor (hMOR) were mutated into alanines and Ala348,353 hMOR was stably expressed in HEK 293 cells. [35S] GTPgammaS binding experiments revealed that Ala348,353 hMOR basal activity was significantly higher when compared to hMOR, suggesting that the mutant receptor is constitutively active. [35S] GTPgammaS binding was decreased by cyprodime or CTOP indicating that both ligands have inverse agonist properties. All tested agonists exhibited binding affinities higher for Ala348,353 hMOR than for hMOR, with the exception of endogenous opioid peptides. Antagonist affinity remained virtually unchanged except for CTOP and cyprodime that bound the double mutant with higher affinities. The agonists DAMGO and morphine showed enhanced potency for the Ala348,353 hMOR receptor in [35S] GTPgammaS experiments. Finally, pretreatment with the antagonists naloxone, cyprodime or CTOP significantly increased Ala348,353 hMOR expression.

Conclusion: Taken together our data indicate that the double C348/353A mutation results in a constitutively active conformation of hMOR that is still activated by agonists. This is the first report of a stable CAM of hMOR with the potential to screen for inverse agonists.

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Figures

Figure 1
Figure 1
Comparison of hMOR and Ala348,353 hMOR [35S] GTPγS binding with or without PTX treatment. Basal (panel A) and DAMGO-induced (panel B) [35S] GTPγS incorporation were measured as described in the Experimental Procedures. Membranes were prepared from HEK 293 cells stably expressing hMOR or Ala348,353 hMOR. Cells were treated with 100 ng/mL PTX for 20 h where indicated. DAMGO was added at a concentration of 10 μM. Data are given as the mean ± S.E.M. from at least 3 independent experiments performed in triplicate. Statistical analysis was performed using Student's t test to evaluate the effects of mutation (***, p < 0.001) or PTX-treatment (##, p < 0.01) on basal [35S] GTPγS incorporation (panel A) and of the effect of PTX-treatment (**, p < 0.01) on DAMGO-induced [35S] GTPγS incorporation (panel B).
Figure 2
Figure 2
DAMGO and morphine-stimulated [35S] GTPγS binding at hMOR and Ala348,353 hMOR. Increasing concentrations of DAMGO and morphine (10-10 to 10-4 M) were used to stimulate [35S] GTPγS binding. Panel A: DAMGO at hMOR (■) or at Ala348,353 hMOR (▼). Panel B: morphine at hMOR (◆) or Ala348,353 hMOR (▲). Data are given as the mean ± S.E.M. from at least 3 independent experiments performed in triplicate.
Figure 3
Figure 3
Effect of antagonists on the incorporation of [35S] GTPγS binding in membranes expressing hMOR and Ala348,353 hMOR. [35S] GTPγS incorporation was measured as described in the Experimental Procedures. Panel A: membranes prepared from HEK 293 cells stably expressing hMOR. Panel B: membranes prepared from HEK 293 cells stably expressing Ala348,353 hMOR. All ligands were used at a concentration of 10 μM. Incorporation is expressed as percent variation over [35S] GTPγS binding in the absence of agonist. Data are given as the mean ± S.E.M. from 3 (except naloxone, CTOP and cyprodime where n = 5) independent experiments performed in triplicate. Statistical analysis was performed using Student's t test, *p < 0.05, **p < 0.01 compared to basal [35S] GTPγS.
Figure 4
Figure 4
Upregulation of hMOR and Ala348,353 hMOR expression upon ligand pretreatment of the cells. Saturation experiments using [3H]diprenorphine on intact HEK 293 cells stably expressing hMOR (checked bars) or Ala348,353 hMOR (black bars) following cell treatment with naloxone, cyprodime or CTOP 1 mM for 48 h as described in the Experimental Procedures. Data are given as the mean ± S.E.M. from at least 4 independent experiments. Statistical analysis was performed using Student's t test to compare upregulation at wild-type and mutant receptors, ** p < 0.01 compared to hMOR.
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