Remember that neither Ser363, Thr370 nor Ser375 phosphorylation was detectable in the Ser363A/Thr370A/Ser375A mutant. indicating that Ser375 is the main site of agonist-dependent phosphorylation. Activation of PKC by phorbol 12-myristate 13-acetate improved receptor phosphorylation only on Thr370, but not on Ser375, indicating that Thr370 can also undergo heterologous PKC-mediated phosphorylation. We also showed that receptor dephosphorylation can occur within minutes at or near the plasma membrane, and that agonist removal is definitely a major prerequisite for Thr370 and Ser375 dephosphorylation. CONCLUSIONS AND IMPLICATIONS Together, we showed for the first time that unique agonists stimulate site-specific patterns of phosphorylation, which are intimately related to their ability to elicit -opioid receptor sequestration. LINKED ARTICLE This short article is definitely commented on by Kelly, pp. 294C297 of this issue. To view this commentary check out http://dx.doi.org/10.1111/j.1476-5381.2011.01387.x strong class=”kwd-title” Keywords: opioid receptor, morphine, tolerance, phosphorylation Intro The opioid alkaloid morphine is among the most potent clinically used analgesic. However, the clinical energy of morphine to treat chronic pain is limited by its quick development of tolerance and dependence (Nestler, 1996; Nestler and Aghajanian, 1997; Koob em et al /em ., 1998). Morphine exerts all of its biological effects by interacting with the -opioid receptor (Matthes em et al /em ., 1996). Like endogenous opioid peptides, morphine binds and activates the receptor (Arden em et al /em ., 1995; Keith em et al /em 7-Epi 10-Desacetyl Paclitaxel ., 1996; Burd em et al /em ., 1998; Koch em et al /em ., 2001). Unlike endogenous opioids, however, morphine does not elicit powerful -opioid receptor sequestration (Arden em et al /em ., 1995; Keith em et al /em ., 1996; Schulz em et al /em ., 2004; Johnson em et al /em ., 2006; McPherson em et al /em ., 2010). To day, the molecular basis for this agonist-selective -opioid receptor internalization remains unknown. Several studies possess reported the crucial part of C-terminal phosphorylation sites in regulating opioid receptor activity and trafficking. Further studies exposed that the ability of unique opioid agonists to differentially regulate receptor endocytosis is related 7-Epi 10-Desacetyl Paclitaxel to their ability to promote GPCR kinase 2 (GRK2)-dependent phosphorylation of the -opioid receptor (Zhang em et al /em ., 1998; Ferguson, 2001; Schulz em et al /em ., 2004; Kenski em et al /em ., 2005). Analysis of serial truncation and site-directed mutants have suggested that phosphorylation of receptors happens primarily at a cluster of three serine and threonine residues, namely serine 363 (Ser363), threonine 370 (Thr370) and serine 375 (Ser375), within the cytoplasmic tail of the receptor (El Kouhen em et al /em ., 2001; Chu em et al /em ., 2008). However, these three sites seem to be phosphorylated in a different way: Ser363 is definitely phosphorylated only 7-Epi 10-Desacetyl Paclitaxel in the basal condition, Thr370 is definitely phosphorylated both in the presence and absence of [d-Ala2-MePhe4-Gly-ol]enkephalin (DAMGO), and Ser375 is definitely phosphorylated only in the presence of DAMGO (El Kouhen em et al /em ., 2001). In support of these findings, it has recently been shown that Ser375 is definitely phosphorylated by GRK2 in an agonist-dependent manner and seems to be important for the induction of opioid receptor endocytosis (Schulz em et al /em ., 2004; McPherson em et 7-Epi 10-Desacetyl Paclitaxel al /em ., 2010). So far, the kinase involved in Thr370 phosphorylation TRAILR4 and its part in the rules of opioid receptor trafficking is definitely unknown. Therefore, in the present study, we have generated and extensively characterized phosphosite-specific antibodies, which allowed us to selectively detect the Ser363-, Thr370- and Ser375-phosphorylated forms of the receptor. Using these antibodies, we provide evidence for unique agonist-selective patterns of -opioid receptor phosphorylation following activation by internalizing or non-internalizing agonists. Methods Animal care and methods All animal experiments were authorized by the Thuringian state government bodies and complied with EC regulations (86/609/EEC) for the care and reporting requirements for use of laboratory animals. Antibodies and reagents Phosphosite-specific antibodies for the Ser363-phosphorylated form of the receptor were generated against the following sequence that 7-Epi 10-Desacetyl Paclitaxel contained a phosphorylated serine residue: EQQN(pS)ARIRQ. This sequence corresponds to amino acids 359C368 of the mouse, and 361C370 of the human being -opioid receptor, respectively. Phosphosite-specific antibodies for the Thr370-phosphorylated form of the receptor were generated against the following sequence that contained a phosphorylated threonine residue: IRQN(pT)REHP. This sequence corresponds to amino acids 366C374 of the mouse, and 368C376 of the human being -opioid receptor, respectively. Phosphosite-specific antibodies for the Ser375-phosphorylated form of the .