Abstract
Chronic opioid treatment of Neuro2A cells stably expressing either δ-opioid receptor (DOR) or μ-opioid receptor (MOR) resulted in agonist-dependent receptor down-regulation. Although there is high homology in the DOR and MOR amino acid sequences, there is an apparent difference in the regulation of the cellular levels of these two receptors. The ability of 24-hr [d-Pen2,d-Pen5]enkephalin (DPDPE) treatment to internalize and down-regulate DORs expressed in Neuro2A remained intact after pertussis toxin (PTX) pretreatment, which uncouples the receptor from G proteins. In contrast, the ability of [d-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO) to internalize and down-regulate MORs in Neuro2Acells was completely abolished by PTX pretreatment. The requirement of functional MOR but not DOR in agonist-induced receptor down-regulation was further demonstrated by site-directed mutagenesis of the receptors. When Asp114 in transmembrane 2 of MOR was converted to alanine, the ability was abolished of DAMGO or morphine to inhibit forskolin-stimulated [3H]cAMP production in Neuro2A cells stably expressing this mutant receptor. There was a parallel decrease in agonist affinity and elimination of the agonist-induced receptor down-regulation. On the other hand, although the equivalent mutation of Asp95 to alanine in DOR likewise resulted in the inability of DPDPE to inhibit [3H]cAMP production, the ability of DPDPE to down-regulate this mutant receptor after 24-hr treatment was unaffected. This difference in MOR and DOR down-regulation could be caused by the differences in the ability of these two receptors to form high affinity complexes with G proteins. DOR retained the ability to form high affinity complexes even after PTX pretreatment or after mutation of Asp95 in transmembrane 2. In contrast, MOR existed only in the low affinity, uncoupled state after PTX pretreatment or after conversion of Asp114 to alanine. Therefore, in Neuro2A cells, agonist-induced opioid receptor down-regulation seems to depend directly on the formation of the high affinity receptor complexes and not on the activation of the receptors and subsequent transduction of the signals.
Footnotes
- Received December 13, 1996.
- Accepted April 4, 1997.
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Send reprint requests to: Dr. S. Chakrabarti, Department of Biochemistry, Box 8, State University of New York, Health Sciences Center at Brooklyn, 450 Clarkson Ave., Brooklyn, NY, 11203-2098. E-mail: schakrabarti{at}netmail.uscbklyn.edu
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↵1 Current affiliation: Department of Biochemistry, State University of New York, Brooklyn, NY 11203-2098.
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This research was supported in parts by Grants DA0564, DA01583, DA5695, and DA07339 and Research Scientist Award K05-DA70554 from the National Institute on Drug Abuse.
- The American Society for Pharmacology and Experimental Therapeutics
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