title: Agonist-selective patterns of µ-opioid receptor phosphorylation revealed by phosphosite-specific antibodies.
abstract: OBJECTIVE
  Morphine activates the µ-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.
  
  
  METHODS
  Here, 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.
  
  
  RESULTS
  We 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 µ 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
  Together, we showed for the first time that distinct agonists stimulate site-specific patterns of phosphorylation, which are intimately related to their ability to elicit µ-opioid receptor sequestration.
  
  
  BACKGROUND
  This article is commented on by Kelly, pp. 294-297 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01387.x.
authors: Institute of Pharmacology and Toxicology, University Hospital, Friedrich Schiller University, Jena, Germany.
authors: Christian
authors: C
authors: Doll
authors: Jens
authors: J
authors: Konietzko
authors: Florian
authors: F
authors: Pöll
authors: Thomas
authors: T
authors: Koch
authors: Volker
authors: V
authors: Höllt
authors: Stefan
authors: S
authors: Schulz
mesh_terms: D000595:Amino Acid Sequence; D000701:Analgesics, Opioid / Q000494:pharmacology*; D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000378:metabolism*; D001562:Benzimidazoles / Q000494:pharmacology; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D009020:Morphine / Q000494:pharmacology; D010766:Phosphorylation; D011817:Rabbits; D017450:Receptors, Opioid, mu / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000701:Analgesics, Opioid; D045163:Antibodies, Phospho-Specific; D001562:Benzimidazoles; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009020:Morphine; C084835:etonitazene