Yousuf Arsalan, Miess Elke, Sianati Setareh, Du Yan-Ping, Schulz Stefan, Christie MacDonald J
Discipline of Pharmacology, University of Sydney, New South Wales, Sydney, Australia (A.Y., S.Si., Y.-P.D., M.J.C.); and Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, Jena, Germany (E.M., S.Sc.).
Discipline of Pharmacology, University of Sydney, New South Wales, Sydney, Australia (A.Y., S.Si., Y.-P.D., M.J.C.); and Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, Jena, Germany (E.M., S.Sc.)
Mol Pharmacol. 2015 Oct;88(4):825-35. doi: 10.1124/mol.115.098244. Epub 2015 May 12.
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 Met(5)-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.
μ-阿片受体（MOPr）C末端尾巴残基的磷酸化被认为是脱敏和内化的关键步骤。C末端S/T残基的磷酸化是内化所必需的（Just等人，2013年），但其在脱敏中的作用尚不清楚。本研究考察了C末端磷酸化位点对MOPr快速脱敏的影响。野生型MOPr、维持内化的3S/T-A突变体（S363A、T370A、S375A）、6S/T-A（S363A、T370A、S375A、T376A、T379A、T383A）和未被MOPr激动剂内化的11S/T-A（所有C末端S/T残基均发生突变）突变体在AtT20细胞中稳定表达。通过穿孔膜片钳记录MOPr介导的由亚最大浓度的Met(5)-脑啡肽（ME）和生长抑素（SST；与天然SST受体[SSTR]偶联）激活G蛋白激活的内向整流钾通道（Kir3.X）（GIRK）电导，以检测在37°C下暴露于ME和吗啡5分钟诱导的脱敏。使用磷酸化位点特异性抗体检测发现，ME和吗啡诱导的脱敏速率与磷酸化无关。ME诱导的MOPr脱敏和再敏化在3S/T-A和6S/T-A中与野生型无差异，但在11S/T-A中被消除。吗啡诱导的脱敏在所有三个突变体中均未受影响，SSTR的异源脱敏也是如此。在野生型MOPr中，蛋白激酶C抑制可降低吗啡诱导的脱敏（但不影响ME诱导的脱敏），在11S/T-A突变体中则被消除，异源脱敏也是如此。这些发现表明，MOPr脱敏可以独立于S/T磷酸化和内化而发生；然而，C末端磷酸化对于某些形式的脱敏是必要的，因为所有C末端位点的突变（11S/T-A）会消除ME诱导的脱敏。
