MOLECULAR MECHANISM OF OPIOID RECEPTOR REGULATION

阿片受体属于G蛋白偶联受体超家族 （GPCR）。类似于大多数这个超家族成员， 阿片受体的激活导致反应的丧失，主要是 因为受体脱敏。有压倒性的证据表明 GPCR的磷酸化是受体的一般机制， 脱敏在阿片受体的情况下， 已经报道了激动剂激活后的μ-δ-阿片样物质受体。 尽管有一些外围迹象表明 δ-阿片受体磷酸化和受体脱敏， 详细的相关性尚未建立。在我们的研究中， 阿片受体磷酸化，我们可以证明， 磷酸化发生在激动剂结合的几分钟内，而 激动剂抑制腺苷酸环化酶的能力直到数小时才减弱 加入激动剂后。因此，我们决定彻底调查 δ-阿片受体磷酸化与 脱敏我们将利用多克隆抗体 针对δ-阿片受体和血凝素（HA）表位标记的 我们在研究中开发的受体。我们将把 δ阿片受体磷酸化/去磷酸化的能力 抑制毛喉素刺激的腺苷酸环化酶活性的激动剂。 我们将研究各种蛋白激酶抑制剂对 受体磷酸化和脱敏。我们将精确定位 δ-阿片受体上的磷酸化位点， 受体脱敏这将由受体完成 截短和突变分析。受体衰减 去除假定磷酸化位点的磷酸化 和Thr，以及随后对激动剂诱导的受体 将确定脱敏。以消除任何 错误的结论，对目的Ser/Thr突变为Asp的影响将是 评估和相关受体结构域的氨基酸序列 将进行磷酸化。最后，蛋白激酶 参与阿片受体磷酸化的蛋白质将通过 这些激酶在HEK 293细胞中的瞬时表达， 表达δ-阿片受体。过度表达的影响 这些激酶，或GRK的显性突变体对受体磷酸化 并将确定脱敏。一个可能存在的 δ-阿片受体的特异性激酶将由免疫组化研究。 磷酸化纯化蛋白的体外磷酸化和肽图谱 受体与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.