Agonist-Selective Mechanisms of μ-Opioid Receptor Desensitization in Human Embryonic Kidney 293 Cells

Abstract

The ability of two opioid agonists, [D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]-enkephalin (DAMGO) and morphine, to induce -opioid receptor (MOR) phosphorylation, desensitization, and internalization was examined in human embryonic kidney (HEK) 293 cells expressing rat MOR1 as well G protein-coupled inwardly rectifying potassium channel (GIRK) channel subunits. Both DAMGO and morphine activated GIRK currents, but the maximum response to DAMGO was greater than that of morphine, indicating that morphine is a partial agonist. The responses to DAMGO and morphine desensitized rapidly in the presence of either drug. Expression of a dominant negative mutant G protein-coupled receptor kinase 2 (GRK2), GRK2-K220R, markedly attenuated the DAMGO-induced desensitization of MOR1, but it had no effect on morphine-induced MOR1 desensitization. In contrast, inhibition of protein kinase C (PKC) either by the PKC inhibitory peptide PKC (19-31) or staurosporine reduced MOR1 desensitization by morphine but not that induced by DAMGO. Morphine and DAMGO enhanced MOR1 phosphorylation over basal. The PKC inhibitor bisindolylmaleimide 1 (GF109203X) inhibited MOR1 phosphorylation under basal conditions and in the presence of morphine, but it did not inhibit DAMGO-induced phosphorylation. DAMGO induced arrestin-2 translocation to the plasma membrane and considerable MOR1 internalization, whereas morphine did not induce arrestin-2 translocation and induced very little MOR1 internalization. Thus, DAMGO and morphine each induce desensitization of MOR1 signaling in HEK293 cells but by different molecular mechanisms; DAMGO-induced desensitization is GRK2-dependent, whereas morphine-induced desensitization is in part PKC-dependent. MORs desensitized by DAMGO activation are then readily internalized by an arrestin-dependent mechanism, whereas those desensitized by morphine are not. These data suggest that opioid agonists induce different conformations of the MOR that are susceptible to different desensitizing and internalization processes.

Involvement of NOX1/NADPH Oxidase in Morphine-Induced Analgesia and Tolerance

“…These findings indicate that morphine elicits desensitization without an apparent change in the number of MOR. This is in agreement with previous studies, which found that morphineinduced desensitization took place without ␤-arrestin-mediated MOR internalization (Johnson et al, 2006;Groer et al, 2007). Most relevantly, it was demonstrated that the initial exposure to morphine causes G␣ depletion due to the interaction of G␣ with RGS, while further exposure to morphine elicits phosphorylation and internalization of MOR (Rodríguez-Muñoz et al, 2007).…”

“…While the molecular mechanisms underlying the development of opioid-induced tolerance have not been fully clarified, desensitization appears to contribute to tolerance. Morphine desensitizes the receptor via a protein kinase C (PKC)-dependent pathway (Johnson et al, 2006), while a synthetic opioid agonist desensitizes MOR -opioid receptor by G-protein-coupled receptor kinase (GRK) dependent internalization and the resultant downregulation of MOR expression (Zhang et al, 1998). Regulator of G-protein signaling (RGS) proteins were identified as taking part in the receptor's desensitization independent of its internalization and downregulation (Garzó n et al, 2001).…”

Two Distinct Mechanisms Mediate Acute μ-Opioid Receptor Desensitization in Native Neurons

“…In addition to GRK-␤arr dependent mechanisms, protein kinase C (PKC) and ERK1/2 activation can mediate or enhance MOR desensitization in heterologous expression systems (Schmidt et al, 2000;Bohn et al, 2002;Johnson et al, 2006). Together, these observations suggest either that MOR desensitization under different experimental conditions can occur via different molecular mechanisms Johnson et al, 2006) or, alternatively, MOR desensitization is mediated by multiple mechanisms in native neurons.…”

“…Together, these observations suggest either that MOR desensitization under different experimental conditions can occur via different molecular mechanisms Johnson et al, 2006) or, alternatively, MOR desensitization is mediated by multiple mechanisms in native neurons. To determine whether MOR desensitization is mediated by multiple molecular mechanisms, this study used patch-clamp recordings of the activation of GIRK channels by MOR in brain slices containing LC neurons from ␤arr-2 wild type (w.t.)…”

A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

“…The hyperpolarization produced by high concentrations of morphine or DAMGO appeared to wane over time, and when the cells were challenged with a concentration of agonist that should saturate the cell surface receptors, there was a marked decrease in this response after only a few minutes of exposure to the agonist, consistent with analogous studies performed in locus coeruleus neurons 27 or cell lines transfected with µ-opioid receptors and GIRK channels. 28 We were able to make use of the endogenous sst receptors in the AtT-20 cells to determine whether exposure to desensitizing concentrations of opioid agonist affected signaling through other receptors. The time constant for the desensitization of signaling produced by morphine was slower than that reported in our previous study of opioid signaling, which used inhibition of I Ca in AtT-20 cells, and this may reflect a distinct recruitment of desensitization processes by morphine in intact cells compared with cells dialyzed by patch-clamp recording.…”