Abstract
Chronic opioid treatment of Neuro2A cells stably expressing either delta-opioid receptor (DOR) or mu-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 Neuro2A cells 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.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.