MOLECULAR MECHANISM OF OPIOID RECEPTOR REGULATION

阿片受体属于G蛋白偶联受体超家族 (GPCRs)。与这个超级家庭的大多数成员相似，延长了 阿片受体的激活导致反应丧失，主要是 由于受体脱敏。有压倒性的证据表明 GPCRs的磷酸化是受体的一般机制 脱敏。在阿片受体的情况下，磷酸化的 β-阿片受体在激动剂激活中的作用已有报道。 尽管有一些外围迹象表明 阿片受体磷酸化和受体脱敏， 详细的相关性尚未建立。在我们与穆的研究中- 阿片受体磷酸化，我们可以证明受体 激动剂结合后几分钟内发生磷酸化，而 激动剂抑制腺酰环化酶的能力直到几个小时才被钝化 加入激动剂后。因此，我们决定彻底调查 β-阿片受体磷酸化与细胞周期调控的关系 脱敏。我们将利用特异性的多克隆抗体 抗标记的阿片受体和血凝素(HA)表位 我们在研究中开发的受体。我们将相关的程度 Delta阿片受体的磷酸化/去磷酸化对 激动剂抑制Forsklin刺激的腺酰环化酶活性。 我们将研究不同的蛋白激酶抑制剂对血管生成的影响。 受体的磷酸化和脱敏。我们将重点指出 β-阿片受体上的磷酸化位点参与 受体脱敏。这将由受体来完成 截断和突变分析。受体的衰减性 去掉假定的磷酸化位点的磷酸化 和苏氨酸，以及随后对激动剂诱导的受体的影响 脱敏作用将被确定。为了消除任何 误判，对Asp感兴趣的Ser/Thr突变的影响将是 相关受体结构域的评估和氨基酸序列分析 将进行磷酸化。最后，它的蛋白激酶 参与阿片受体磷酸化的RE将通过 这些蛋白在HEK293细胞中的瞬时表达 表达阿片受体。基因过度表达的影响 这些激酶，或受体磷酸化GRK的显性突变体 脱敏作用将被确定。很可能出现了一个 三角洲阿片受体的特定激酶将由IN进行研究 磷酸化纯化产物的体外磷酸化及肽谱分析 受体通过内源性和外源性蛋白激酶进行。

Opioid receptors belong the superfamily of G protein-coupled receptors (GPCRs). Similar to a majority of this superfamily members, prolonged activation of the opioid receptors resulted in a loss of response, mainly due to receptor desensitization. There is overwhelming evidence to suggest that the phosphorylation of GPCRs is the general mechanism for receptor desensitization. In the case of opioid receptor, phosphorylation of the mu- nd delta-opioid receptor upon agonist activation have been reported. Though there is some peripheral indication of a relationship between delta-opioid receptor phosphorylation and receptor desensitization, detailed correlation has not been established. In our studies with mu- opioid receptor phosphorylation, we could demonstrate that receptor phosphorylation occurred within minutes of agonist binding, while the ability of agonist to inhibit adenylyl cyclase was not blunted until hours after agonist addition. Therefore, we decided to investigate thoroughly the relationship between delta-opioid receptor phosphorylation and desensitization. We will utilize the polyclonal antibodies specific against the delta-opioid receptor and hemagglutinin (HA) epitope tagged receptor we have developed in our studies. We will correlate the degree of delta opioid receptor phosphorylation/dephosphorylation to the ability of agonist in inhibiting the forskolin-stimulated adenylyl cyclase activity. We will investigate the effect of various protein kinases' inhibitors on receptors phosphorylation and desensitization. We will pin-point the phosphorylation sites on the delta-opioid receptor which are involved in receptor desensitization. This will be accomplish by the receptor truncational and mutational analysis. The attenuation of receptor phosphorylation with the removal of putative phosphorylation sites, SER and Thr, and the subsequent effect on agonist-induced receptor desensitization will be determined. In order to eliminate any misconclusion, effect on mutation of Ser/Thr of interest to Asp will be evaluated and the amino acid sequencing of the receptor domains involved in phosphorylation will be carried out. Finally, the protein kinases which re involved in opioid receptor phosphorylation will be identified by the transient expression of these kinases in HEK293 cells which stably expressing the delta-opioid receptor. The effect of the over-expression of these kinases,or the dominant mutants of GRK on receptor phosphorylation and desensitization will be determined. The probably presence of a specific kinases for delta-opioid receptor will be investigated by the in vitro phosphorylation and peptide mapping of the phosphorylated purified receptor carried out with endogenous and exogenous protein kinases.