Pak Y, O'Dowd B F, Wang J B, George S R
Department of Pharmacology, University of Toronto, Ontario M5S 1A8, Canada.
J Biol Chem. 1999 Sep 24;274(39):27610-6. doi: 10.1074/jbc.274.39.27610.
The mu opioid receptor (MOR) has been shown to desensitize after 1 h of exposure to the opioid peptide, [D-Ala(2), N-MePhe(4), Gly-ol(5)]enkephalin (DAMGO), largely by the loss of receptors from the cell surface and receptor down-regulation. We have previously shown that the Thr(394) in the carboxyl tail is essential for agonist-induced early desensitization, presumably by serving as a primary phosphorylation site for G protein-coupled receptor kinase. Using a T394A mutant receptor, we determined that Thr(394) was also responsible for mu opioid receptor down-regulation. The T394A mutant receptor displayed 50% reduction of receptor down-regulation (14.8%) compared with wild type receptor (34%) upon 1 h of exposure to DAMGO. Agonist-induced T394A receptor down-regulation was unaffected by pertussis toxin treatment, indicating involvement of a mechanism independent of G protein function. Interestingly, pertussis toxin-insensitive T394A receptor down-regulation was completely inhibited by a tyrosine kinase inhibitor, genistein. Tyrosine kinase inhibition blocked wild type MOR down-regulation by 50%, and the genistein-resistant wild type MOR down-regulation was completely pertussis toxin-sensitive. Following DAMGO stimulation, MOR was shown to be phosphorylated at tyrosine residue(s), indicating that the receptor was a direct substrate for tyrosine kinase action. Mutagenesis of the four intracellular tyrosine residues resulted in complete inhibition of the G protein-insensitive MOR internalization. Therefore, agonist-induced MOR down-regulation appears to be mediated by two distinct cellular signal transduction pathways. One is G protein-dependent and GRK-dependent, which can be abolished by pertussis toxin treatment of wild type MOR or by mutagenesis of Thr(394). The other novel pathway is G protein-independent but tyrosine kinase-dependent, blocked by genistein treatment, and one in which Thr(394) has no regulatory role but phosphorylation of tyrosine residues appears essential.
μ阿片受体（MOR）在暴露于阿片肽[D - 丙氨酸（2），N - 甲基苯丙氨酸（4），甘氨醇（5）]脑啡肽（DAMGO）1小时后会出现脱敏现象，这主要是由于细胞表面受体的丧失和受体下调。我们之前已经表明，羧基末端的苏氨酸（Thr394）对于激动剂诱导的早期脱敏至关重要，推测它作为G蛋白偶联受体激酶的主要磷酸化位点。使用T394A突变受体，我们确定Thr394也与μ阿片受体下调有关。与野生型受体（34%）相比，在暴露于DAMGO 1小时后，T394A突变受体的受体下调减少了50%（降至14.8%）。激动剂诱导的T394A受体下调不受百日咳毒素处理的影响，表明涉及一种独立于G蛋白功能的机制。有趣的是，百日咳毒素不敏感的T394A受体下调被酪氨酸激酶抑制剂染料木黄酮完全抑制。酪氨酸激酶抑制使野生型MOR下调50%，而对染料木黄酮耐药的野生型MOR下调则完全对百日咳毒素敏感。在DAMGO刺激后，MOR显示在酪氨酸残基处被磷酸化，表明该受体是酪氨酸激酶作用的直接底物。对四个细胞内酪氨酸残基进行诱变导致对G蛋白不敏感的MOR内化完全被抑制。因此，激动剂诱导的MOR下调似乎由两种不同的细胞信号转导途径介导。一种是G蛋白依赖性和GRK依赖性的，通过对野生型MOR进行百日咳毒素处理或对Thr394进行诱变可以消除。另一种新途径是G蛋白非依赖性但酪氨酸激酶依赖性的，被染料木黄酮处理阻断，在这种途径中Thr394没有调节作用，但酪氨酸残基的磷酸化似乎至关重要。
