PT  - JOURNAL ARTICLE
AU  - Arsalan Yousuf
AU  - Elke Miess
AU  - Setareh Sianati
AU  - Yan-Ping Du
AU  - Stefan Schulz
AU  - MacDonald J. Christie
TI  - Role of Phosphorylation Sites in Desensitization of <em>µ</em>-Opioid Receptor
AID  - 10.1124/mol.115.098244
DP  - 2015 Oct 01
TA  - Molecular Pharmacology
PG  - 825--835
VI  - 88
IP  - 4
4099  - http://molpharm.aspetjournals.org/content/88/4/825.short
4100  - http://molpharm.aspetjournals.org/content/88/4/825.full
SO  - Mol Pharmacol2015 Oct 01; 88
AB  - Phosphorylation of residues in the C-terminal tail of the µ-opioid receptor (MOPr) is thought to be a key step in desensitization and internalization. Phosphorylation of C-terminal S/T residues is required for internalization (Just et al., 2013), but its role in desensitization is unknown. This study examined the influence of C-terminal phosphorylation sites on rapid desensitization of MOPr. Wild-type MOPr, a 3S/T-A mutant (S363A, T370A, S375A) that maintains internalization, 6S/T-A (S363A, T370A, S375A, T376A, T379A, T383A) and 11S/T-A (all C-terminal S/T residues mutated) mutants not internalized by MOPr agonists were stably expressed in AtT20 cells. Perforated patch-clamp recordings of MOPr-mediated activation of G-protein–activated inwardly rectifying potassium channel (Kir3.X) (GIRK) conductance by submaximal concentrations of Met5-enkephalin (ME) and somatostatin (SST; coupling to native SST receptor [SSTR]) were used to examine desensitization induced by exposure to ME and morphine for 5 minutes at 37°C. The rates of ME- and morphine-induced desensitization did not correlate with phosphorylation using phosphorylation site-specific antibodies. ME-induced MOPr desensitization and resensitization did not differ from wild-type for 3S/T-A and 6S/T-A but was abolished in 11S/T-A. Morphine-induced desensitization was unaffected in all three mutants, as was heterologous desensitization of SSTR. Morphine-induced desensitization (but not ME) was reduced by protein kinase C inhibition in wild-type MOPr and abolished in the 11S/T-A mutant, as was heterologous desensitization. These findings establish that MOPr desensitization can occur independently of S/T phosphorylation and internalization; however, C-terminal phosphorylation is necessary for some forms of desensitization because mutation of all C-terminal sites (11S/T-A) abolishes desensitization induced by ME.