Agonist-selective patterns of µ-opioid receptor phosphorylation revealed by phosphosite-specific antibodies
Version of Record online: 22 AUG 2011
© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society
British Journal of Pharmacology
Volume 164, Issue 2, pages 298–307, September 2011
How to Cite
Doll, C., Konietzko, J., Pöll, F., Koch, T., Höllt, V. and Schulz, S. (2011), Agonist-selective patterns of µ-opioid receptor phosphorylation revealed by phosphosite-specific antibodies. British Journal of Pharmacology, 164: 298–307. doi: 10.1111/j.1476-5381.2011.01382.x
- Issue online: 22 AUG 2011
- Version of Record online: 22 AUG 2011
- Accepted manuscript online: 30 MAR 2011 07:46AM EST
- Received; 20 September 2010; Revised; 11 February 2011; Accepted; 17 February 2011
- opioid receptor;
BACKGROUND AND PURPOSE Morphine activates the µ-opioid receptor without causing its rapid endocytosis. In contrast, full agonists such as [d-Ala2-MePhe4-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 APPROACH 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.
KEY 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 AND IMPLICATIONS 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.
LINKED ARTICLE 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