Desensitization and Trafficking of μ-Opioid Receptors in Locus Ceruleus Neurons: Modulation by Kinases

Abstract

The phosphorylation of -opioid receptors (MOPRs) by G protein-coupled receptor kinases (GRKs), followed by arrestin binding, is thought to be a key pathway leading to desensitization and internalization. The present study used the combination of intracellular and whole-cell recordings from rats and mice, as well as live cell imaging of Flag-tagged MOPRs from mouse locus ceruleus neurons, to examine the role of protein kinases in acute desensitization and receptor trafficking. Inhibition of GRKs by using heparin or GRK2-mutant mice did not block desensitization or alter the rate of recovery from desensitization. The nonselective kinase inhibitor staurosporine did not reduce the extent of [Met 5 ]enkephalin (ME)-induced desensitization but increased the rate of recovery from desensitization. In the presence of staurosporine, ME-activated FlagMOPRs were internalized but did not traffic away from the plasma membrane. The increased rate of recovery from desensitization correlated with the enhancement in the recycling of receptors to the plasma membrane. ME-induced MOPR desensitization persisted and the trafficking of receptors was modified after inhibition of protein kinases. The results suggest that desensitization of MOPRs may be an early step after agonist binding that is modulated by but is not dependent on kinase activity.

Regulation ofµ-Opioid Receptors: Desensitization, Phosphorylation, Internalization, and Tolerance

“…These results are in line with previous findings demonstrating an important role of p38 MAPK activation in the induction of endocytotic receptor trafficking (Cavalli et al, 2001;Macé et al, 2005;McLaughlin et al, 2006;Vergarajauregui et al, 2006). It should be noted, however, that inhibition of p38 MAPK in mouse locus coeruleus neurons blocked neither desensitization nor endocytosis of MOR but did increase the rate of recovery from desensitization (Arttamangkul et al, 2012).…”

“…However, blocking both processes nearly abolished MOR desensitization. Another study testing a combination of kinase inhibitors, including staurosporine and heparin, observed that acute desensitization to ME persisted, although there was a significant qualitative change in the pattern of receptor trafficking (Arttamangkul et al, 2012). The reason for persistence of desensitization in these studies when others examining the same neurons have observed greatly attenuated desensitization is not known.…”

“…It is not certain if phosphorylation events, b-arrestin binding, or both produce uncoupling of MOR signaling (desensitization), but the time course appears to follow arrestin binding more closely. Strong internalizing agonists activate phospholipase D2 (Section V.G), but it is not certain whether this is required for endocytosis (Arttamangkul et al, 2012). There is some evidence that ERK1/2-dependent mechanisms may desensitize MOR by both arrestin-independent (Gbg) and arrestin-dependent mechanisms.…”

Role of G Protein–Coupled Receptor Kinases 2 and 3 inμ-Opioid Receptor Desensitization and Internalization

“…2). Other approaches previously taken to inhibit GRKs include the broad-spectrum kinase inhibitor staurosporine (Arttamangkul et al, 2012), the PKC/GRK inhibitor 7,8,indol-10-yl]-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione hydrochloride] (Hull et al, 2010), "b-ARK-1 inhibitor" (Iino et al, 2002;Hull et al, 2010), and the antidepressant drug paroxetine (Thal et al, 2012;Homan et al, 2014b). However, none of these approaches combine the cell-permeability, potency, and selectivity profiles of Cmpd101.…”

“…On the other hand, two studies have failed to provide evidence for any involvement of GRKs in MOPr desensitization. Neither heparin, a low-affinity GRK inhibitor, nor staurosporine, a nonselective kinase inhibitor, had any effect on methionine-enkephalin (Met-Enk)-induced MOPr desensitization in rat LC neurons (Arttamangkul et al, 2012). Also, using the GRK2as5 transgenic mouse, in which GRK2 has been mutated to allow chemical inhibition, it was observed that exposure to the inhibitor did not reduce Met-Enk-induced desensitization in LC neurons (Quillinan et al, 2011).…”