PHOSPHORYLATIVE DECODING OF OPIATE INTERACTIONS USING MASS SPECTROMETRY

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。μ阿片G蛋白偶联受体的脱敏和内化是由G蛋白偶联受体激酶（GRKs）在胞质尾区的多个丝氨酸和苏氨酸残基上的磷酸化介导的配体依赖性过程。虽然内源性肽和美沙酮诱导磷酸化，然后受体内化到细胞质中，但某些高度成瘾的药物如海洛因和吗啡在磷酸化、脱敏和内化方面的作用显著不同。这些差异中的一些可以根据激动剂功效的经典模型来理解。然而，一些证据表明，阿片类药物的调节作用可能有额外的特异性，目前尚无法解释。所提出的研究的工作假设是，阿片类药物，除了不同的相对功效，促进G蛋白活化，产生不同的模式的多个磷酸化的μ阿片受体，从而“编码”的一些差异，在以前的研究中观察到的细胞调节。拟议的研究将使用先前定义的体外和基于细胞的系统来测试这一假设，以在受控条件下产生磷酸化受体，然后使用先进的蛋白质质谱法来精确定义产生的受体磷酸化模式。然后使用已知对阿片受体调节发生激动剂特异性作用的转染细胞来测试受体磷酸化中推定的激动剂特异性差异的功能意义。拟议的研究可以提供显着的阿片类药物的作用机制的新见解，更一般地说，可能有助于扩大我们目前的理解部分激动的GPCR。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Desensitization and internalization of the mu opioid G protein-coupled receptor are ligand dependent processes mediated by phosphorylation by G protein-coupled receptor kinases (GRKs) on multiple serine and threonine residues in the cytoplasmic tail. While endogenous peptides and methadone induce phosphorylation followed by receptor internalization into the cytoplasm, certain highly addictive drugs such as heroin and morphine differ significantly in their effects on phosphorylation, desensitization, and internalization. Some of these differences can be understood in terms of classical models of agonist efficacy. However, several lines of evidence suggest that there may be additional specificity in the regulatory effects of opiate drugs that are currently unexplained. The working hypothesis of the proposed studies is that opiate drugs, in addition to differing in relative efficacy for promoting G protein activation, produce different patterns of multiple phosphorylations in the mu opioid receptor, thereby 'encoding' some of the differences in cellular regulation observed in previous studies. The proposed studies will test this hypothesis using previously defined in vitro and cell-based systems to generate phosphorylated receptors under controlled conditions, followed by advanced protein mass spectrometry to precisely define patterns of receptor phosphorylation produced. The functional significance of putative agonist-specific differences in receptor phosphorylation will then be tested using transfected cells in which agonist-specific effects on opioid receptor regulation are known to occur. The proposed studies could provide significant new insight into mechanisms of opiate drug action and, more generally, may help extend our present understanding of partial agonism of GPCRs.