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Allelic variation S268P of the human mu-opioid receptor affects both desensitization and G protein coupling
T Koch1, T Kroslak, M Averbeck
1Department of Pharmacology and Toxicology, Otto-von-Guericke University, Magdeburg, Germany.
Abstract:
The decrease in mu-opioid receptor activity after chronic agonist exposure (1 microM [D-Ala(2),N-MePhe(4),Gly-ol(5)]-enkephalin) is largely due to kinase-mediated phosphorylation of intracellular receptor domains. We have recently shown that the substitution of two putative Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) phosphorylation sites, S261 and S266, by alanines in the third intracellular loop of the rat mu-opioid receptor (rMOR1) confers resistance to CaMK II-induced receptor desensitization. In the present study, we show that the injection of active CaMK II in Xenopus laevis oocytes led to the desensitization of S261A but not S266A receptor mutant, indicating that S266 is the primary CaMK II phosphorylation site of the rMOR1. For the corresponding phosphorylation site in the human mu-opioid receptor (hMOR), an allelic variation S268P has been recently identified. After expression in X. laevis oocytes and human embryonic kidney 293 cells, this human S268P receptor and a corresponding rat S266P receptor mutant revealed a loss of CaMK II-induced receptor desensitization and a decreased G protein coupling compared with the wild-type receptors. Our results suggest that serines 266 (rMOR1) and 268 (hMOR) play crucial role in receptor desensitization and signaling and that the allelic variation S268P results in a human receptor type with a weaker but persistent G protein coupling after agonist treatment.
Insights
Chronic mu-opioid receptor (MOR) activation causes desensitization via Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) phosphorylation. Serine 266 in rat MOR and Serine 268 in human MOR are key sites for this process.
Area of Science:
- Pharmacology
- Molecular Biology
- Neuroscience
Background:
- Chronic mu-opioid receptor (MOR) activation leads to decreased receptor activity, primarily through kinase-mediated phosphorylation of intracellular domains.
- Previous work identified Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) phosphorylation sites (S261 and S266) in the rat MOR (rMOR1) third intracellular loop, with alanine substitutions conferring resistance to CaMK II-induced desensitization.
Purpose of the Study:
- To identify the primary CaMK II phosphorylation site on the rMOR1.
- To investigate the functional consequences of a specific human MOR (hMOR) allelic variation (S268P) corresponding to a key rat phosphorylation site.
- To elucidate the role of specific serine residues in MOR desensitization and G protein coupling.
Main Methods:
- Expression of wild-type and mutant rat and human mu-opioid receptors in Xenopus laevis oocytes and human embryonic kidney 293 cells.
- Injection of active CaMK II into oocytes expressing receptor mutants.
- Assessment of receptor desensitization and G protein coupling following agonist stimulation.
Main Results:
- Active CaMK II induced desensitization of the rMOR1 S261A mutant but not the S266A mutant, identifying S266 as the primary CaMK II phosphorylation site.
- The human S268P receptor mutant and the corresponding rat S266P mutant exhibited reduced CaMK II-induced desensitization compared to wild-type receptors.
- Both human S268P and rat S266P mutants showed decreased G protein coupling after agonist treatment.
Conclusions:
- Serines 266 (rMOR1) and 268 (hMOR) are critical for mu-opioid receptor desensitization and signaling.
- The human S268P allelic variation results in a receptor with diminished G protein coupling persistence after agonist exposure, suggesting altered signaling properties.