Agonist-selective patterns of µ-opioid receptor phosphorylation revealed by phosphosite-specific antibodies

Abstract

BACKGROUND AND PURPOSEMorphine activates the m-opioid receptor without causing its rapid endocytosis. In contrast, full agonists such as [D-Ala 2 -MePhe 4 -Gly-ol]enkephalin (DAMGO) or etonitazene stimulate a rapid and profound internalization. However, the detailed molecular events underlying the differential regulation of receptor trafficking by distinct opioid agonists remain incompletely understood. EXPERIMENTAL APPROACHHere, we have generated phosphosite-specific antibodies for the carboxyl-terminal residues serine 363 (Ser363), threonine 370 (Thr370) and serine 375 (Ser375), which enabled us to selectively detect either the Ser363-, Thr370-or Ser375-phosphorylated form of the receptor. KEY RESULTSWe showed that agonist-induced phosphorylation occurs at Thr370 and Ser375, whereas Ser363 is constitutively phosphorylated in the absence of agonist. We further demonstated that DAMGO and etonitazene stimulated the phosphorylation of both Thr370 and Ser375. In contrast, morphine promoted the phosphorylation of Ser375, but failed to stimulate Thr370 phosphorylation. In the presence of DAMGO, Ser375 phosphorylation occurred at a faster rate than phosphorylation of Thr370, indicating that Ser375 is the primary site of agonist-dependent phosphorylation. Activation of PKC by phorbol 12-myristate 13-acetate increased receptor phosphorylation only on Thr370, but not on Ser375, indicating that Thr370 can also undergo heterologous PKC-mediated phosphorylation. We also showed that m receptor dephosphorylation can occur within minutes at or near the plasma membrane, and that agonist removal is a major prerequisite for Thr370 and Ser375 dephosphorylation. CONCLUSIONS AND IMPLICATIONSTogether, we showed for the first time that distinct agonists stimulate site-specific patterns of phosphorylation, which are intimately related to their ability to elicit m-opioid receptor sequestration. LINKED ARTICLE

Analgesic Tolerance to High-Efficacy Agonists But Not to Morphine Is Diminished in Phosphorylation-Deficient S375A μ-Opioid Receptor Knock-In Mice

“…S375 is the primary site of phosphorylation that contributes to desensitization, ␤-arrestin binding, and internalization of the -opioid receptor (El Kouhen et al, 2001;Schulz et al, 2004;McPherson et al, 2010;Kelly, 2011). In contrast, morphine promoted the phosphorylation of S375 but failed to stimulate T370 phosphorylation (Schulz et al, 2004;Doll et al, 2011). Here, we show that the morphine-mediated S375 phosphorylation also occurs in vivo shortly after administration of the drug.…”

“…Upon exposure to DAMGO, S375 is phosphorylated more rapidly than T370. T370 phosphorylation and internalization is strongly reduced in the S375A mutant, suggesting that S375 is the primary site of phosphorylation and that phosphorylation of T370 is in part dependent on the prior phosphorylation of S375 (Schulz et al, 2004;Doll et al, 2011).…”

“…This sequence corresponds to amino acids 371-380 of the mouse -opioid receptor. The anti-pS375 guinea pig polyclonal anti-MOR antibody {GP2} has been generated and characterized in an identical manner to that previously described for the anti-pS375 rabbit polyclonal anti-MOR antibody {2493} (Doll et al, 2011b). When indicated, lysates were sequentially incubated with WGA (wheat germ lectin agarose) beads to enrich glycoproteins after UMB-3 immunoprecipitation.…”

“…To facilitate detection of endogenous -opioid receptors in mouse brain, we have extensively characterized the rabbit monoclonal anti-MOR antibody {UMB-3} (Doll et al, 2011b;Lupp et al, 2011). When crude brain homogenates from MOR ϩ/ϩ mice were immunoprecipitated with UMB-3, the guinea pig anti-MOR antibody {GP6} detected a broad band migrating at M r 70,000 -80,000 in the subsequent immunoblot (Fig.…”

GRK2 Protein-mediated Transphosphorylation Contributes to Loss of Function of μ-Opioid Receptors Induced by Neuropeptide FF (NPFF2) Receptors

“…Previous studies have shown that Thr-370 and Ser-375 are the main targets of homologous regulation by G protein-coupled receptor kinases in the C-terminal tail of the rodent MOP receptor (35)(36)(37)(38), although a potential role for Thr-180 in the second intracellular loop has also been suggested in Xenopus oocytes (39). Here, we have studied the phosphorylation status of the human MOP receptor after homologous stimulation by its agonist (DAMGO) or after heterologous regulation by NPFF 2 receptors.…”