RT Journal Article
SR Electronic
T1 Role of Phosphorylation Sites in Desensitization of <em>µ</em>-Opioid Receptor
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 825
OP 835
DO 10.1124/mol.115.098244
VO 88
IS 4
A1 Arsalan Yousuf
A1 Elke Miess
A1 Setareh Sianati
A1 Yan-Ping Du
A1 Stefan Schulz
A1 MacDonald J. Christie
YR 2015
UL http://molpharm.aspetjournals.org/content/88/4/825.abstract
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.