PHOSPHORYLATIVE DECODING OF OPIATE INTERACTIONS USING MASS SPECTROMETRY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 Mu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)在胞质尾部多个丝氨酸和苏氨酸残基上的磷酸化所介导的配体依赖的过程。虽然内源性多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明显不同。其中一些差异可以用激动剂疗效的经典模型来理解。然而，几条证据表明，阿片类药物的调节效应可能有额外的特异性，目前尚不清楚。拟议研究的工作假设是，阿片类药物除了在促进G蛋白激活方面的相对疗效不同外，还会在Mu阿片受体中产生不同的多重磷酸化模式，从而对先前研究中观察到的细胞调控中的一些差异进行编码。拟议的研究将使用先前定义的体外和基于细胞的系统来验证这一假设，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仪来精确定义产生的受体磷酸化模式。然后，将使用已知发生激动剂对阿片受体调节的激动剂特定效应的转基因细胞来测试假定的激动剂特定的受体磷酸化差异的功能意义。拟议的研究可以为阿片类药物的作用机制提供重要的新见解，更广泛地说，可能有助于扩大我们目前对GPCRs部分兴奋的理解。

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.