Differences in the ability of opioid drugs to promote regulated endocytosis of m-opioid receptors are related to their tendency to produce drug tolerance and dependence. Here we show that drugspecific differences in receptor internalization are determined by a conserved, 10-residue sequence in the receptor's carboxylterminal cytoplasmic tail. Diverse opioids induce receptor phosphorylation at serine (S)375, present in the middle of this sequence, but opioids differ markedly in their ability to drive higher-order phosphorylation on flanking residues [threonine (T)370, T376, and T379]. Multi-phosphorylation is required for the endocytosispromoting activity of this sequence and occurs both sequentially and hierarchically, with S375 representing the initiating site. Higherorder phosphorylation involving T370, T376, and T379 specifically requires GRK2/3 isoforms, and the same sequence controls opioid receptor internalization in neurons. These results reveal a biochemical mechanism differentiating the endocytic activity of opioid drugs.

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

BACKGROUND AND PURPOSEThe molecular basis of agonist-selective signalling at the m-opioid receptor is poorly understood. We have recently shown that full agonists such as [D-Ala ), and that is followed by a robust receptor internalization. In contrast, morphine promotes a selective phosphorylation of Ser 375 without causing rapid receptor internalization. EXPERIMENTAL APPROACHHere, we identify kinases and phosphatases that mediate agonist-dependent phosphorylation and dephosphorylation of the m-opioid receptor using a combination of phosphosite-specific antibodies and siRNA knock-down screening in HEK293 cells. KEY RESULTSWe found that DAMGO-driven phosphorylation of Thr 370 and Ser 375 was preferentially catalysed by G-protein-coupled receptor kinases (GRKs) 2 and 3, whereas morphine-driven Ser 375 phosphorylation was preferentially catalysed by GRK5. On the functional level, inhibition of GRK expression resulted in enhanced m-opioid receptor signalling and reduced receptor internalization. Analysis of GRK5-deficient mice revealed that GRK5 selectively contributes to morphine-induced Ser 375 phosphorylation in brain tissue. We also identified protein phosphatase 1g as a m-opioid receptor phosphatase that catalysed Thr 370 and Ser 375 dephosphorylation at or near the plasma membrane within minutes after agonist removal, which in turn facilitates receptor recycling. CONCLUSIONS AND IMPLICATIONSTogether, the morphine-activated m-opioid receptor is a good substrate for phosphorylation by GRK5 but a poor substrate for GRK2/3. GRK5 phosphorylates m-opioid receptors selectively on Ser 375 , which is not sufficient to drive significant receptor internalization.

Thus, the rabbit monoclonal antibody UMB-1 may prove of great value in the assessment of sst2A status in human neuroendocrine tumors during routine histopathological examination.