Tyrosine Phosphorylation of the κ-Opioid Receptor Regulates Agonist Efficacy

Abstract

To explore the role of highly conserved tyrosine residues in the putative cytoplasmic domains of the seven-transmembrane G protein-coupled opioid receptors, we expressed the rat -opioid receptor (KOR) in Xenopus oocytes and then activated the intrinsic insulin receptor tyrosine kinase. KOR activation by the agonist U69593 produced a strong increase in potassium current through coexpressed G protein-gated inwardly rectifying potassium channels (K IR 3). Brief pretreatment with insulin caused a 60% potentiation of the KOR-activated response. The insulin-induced increase in -opioid response was blocked by the tyrosine kinase inhibitor genistein. In contrast, insulin had no effect on the basal activity of K IR 3, suggesting that KOR is the target of the tyrosine kinase cascade. Mutation of tyrosine residues to phenylalanines in either the first or second intracellular loop of KOR to produce KOR(Y87F) and KOR(Y157F) had no effect on either the potency or maximal effect of U69593. However, neither KOR(Y87F)-nor KOR(Y157F)-mediated responses were potentiated by insulin treatment. Insulin pretreatment shifted the dose-response curve for U69593 activation of KOR by increasing the maximal response without changing the EC 50 value for U69593. These results suggest that insulin increases the efficacy of KOR activation by phosphorylating two tyrosine residues in the first and second intracellular loops of the receptor. Thus, tyrosine phosphorylation may provide an important mechanism for modulation of G protein-coupled receptor signaling.Opioid receptors are widely expressed throughout the nervous system, and opioid drugs affect pain perception, learning and memory, epilepsy, and food intake, among other diverse functions (1). A common mechanism for post-translational regulation of protein function is by phosphorylation, and phosphorylation of opioid receptors is likely to regulate opioid actions. For example, phosphorylation by G protein receptor kinases following agonist treatment has been proposed to play a role in opioid receptor desensitization and tolerance (2). Other serine/ threonine kinases such as protein kinase C and calcium/calmodulin-dependent kinase II in some systems may also regulate opioid receptors (3).To date, few studies have reported any effects of tyrosine kinases on acute opioid receptor function. Tyrosine residues in the putative cytoplasmic domains of G protein-coupled receptors are extremely common, and regulation of opioid receptor signaling by tyrosine kinase cascades would provide a powerful mechanism of cellular coordination. Indeed, a portion of the agonist-induced internalization of the -opioid receptor is dependent on tyrosine kinase activation (4 -6). Opioid receptor activation has been shown to increase tyrosine kinase activity (7,8), and the agonist-induced internalization mediated by tyrosine kinase could potentially involve a process that is directly activated by opioid receptors. These findings are consistent with data on the ␤ 2 -adrenergic receptor in which tyrosine phosphorylation of...

Phosphorylation of the μ-Opioid Receptor at Tyrosine 166 (Tyr3.51) in the DRY Motif Reduces Agonist Efficacy

“…In addition, MOR contains four highly conserved cytoplasmic tyrosine residues (Thompson et al, 1993), and tyrosine kinase-mediated mechanisms regulating MOR signaling have also been described previously (McLaughlin and Chavkin, 2001;Zhang et al, 2009). Tyrosine phosphorylation may influence MOR trafficking and signaling (Pak et al, 1999), consistent with the effects of tyrosine phosphorylation on internalization and signaling of the ␦-and -opioid receptors (Kramer et al, 2000;Appleyard et al, 2000). A recent study by Law and colleagues showed that tyrosine phosphorylation of MOR at Tyr166 and Tyr336 controlled the switch from inhibition to stimulation of adenylyl cyclase after prolonged agonist application (Zhang et al, 2009).…”

Opioid Receptors

“…Opioid receptors can also be tyrosine phosphorylated. This modification affects desensitization as well, as has been shown in a series of studies examining C-terminal residues (Tyr96, Tyr106, Tyr166, and Tyr336) in the MOP-R (140), Tyr318 in the DOP-R (141,142), and residues in the first and second intracellular loop of the KOP-R (143). The net amount of receptor desensitization is thus controlled by many factors, which include phosphorylation, dephosphorylation, new receptor protein synthesis and secretion, as well as receptor endocytosis, recycling, and degradation (see below).…”

Butorphanol: Effects of a Prototypical Agonist-Antagonist Analgesic on κ-Opioid Receptors

“…Phosphorylation of these residues has varying effects on the affinity of the protein for the GTP-bound form of G-proteins and modulates κ-opioid receptor signaling efficiency (30). Another important phosphorylation site is a serine residue (Ser 369 ) on the C-terminus.…”