Receptor Structural Features Determining Drug Tolerance

药物耐受是理解药物成瘾的关键问题，在慢性疾病的治疗中具有重要意义。G蛋白偶联受体（GPCR）是非法药物最重要的细胞靶点之一，并负责当今市场上约60%药物的作用。生化数据表明，这些受体的C-末端对药物脱敏并在细胞内内化。这些过程与药物耐受性的发生有关。人们认为，GPCR C末端的磷酸化以及由此产生的与β-抑制蛋白（一种细胞质调节蛋白）的相互作用决定了这些受体激活后的命运。关于这些事件如何介导的精确结构细节尚不清楚。大麻素受体（CB1）是一种GPCR，负责δ 9-四氢大麻酚（大麻的活性成分）的生物学效应。这种受体作为一个很好的模型，这些机制的结构特性的调查。生物化学证据导致了这样的假设，即CB1的C-末端能够与β-抑制蛋白形成不同的复合物，交替复合物仅导致脱敏，仅导致内化，或两者兼而有之。该项目是一项试点研究，其中精心设计的肽的核磁共振光谱将用于解决以下具体目标i）确定CB 1的C-末端的结构模型ii）测量磷酸化对结构的影响iii）确定适当的条件并测量肽与β-抑制蛋白结合的动力学。该项目的长期目标是获得arrestin和受体之间相互作用的精确知识，这是了解耐受性分子细节的第一步。这些知识将有益于人类健康，为设计配体提供基础，这些配体选择性地防止脱敏，同时允许内化，或者相反地，防止内化，同时允许脱敏。这样的配体将是有价值的生物学工具，在评估这些过程如何有助于药物成瘾。此外，这些信息可能有助于设计规避耐受性的治疗药物。

Drug tolerance is a key issue in understanding drug addiction and is of great importance in the treatment of chronic diseases. G-protein coupled receptors (GPCRs) are among the most important cellular targets for illicit drugs and are responsible for the action of approximately 60% of drugs on the market today. Biochemical data have implicated the C-terminus of these receptors with desensitization towards the drug and internalization within the cell. These processes have been implicated with the onset of drug tolerance. It is thought that phosphorylation of the C- terminus of GPCRs and the resulting interaction with beta-arrestin, a cytosolic regulatory protein, governs the fate of these receptors following activation. The precise structural detail as to how these events are mediated is not known. The cannabinoid receptor (CB1) is a GPCR responsible for the biological effects of delta 9-tetrahydrocannabinol, the active constituent of marijuana. This receptor serves as an excellent model for the investigation of structural properties underlying these mechanisms. The biochemical evidence leads to the hypothesis that the C-terminus of CB1 is able to form differing complexes with beta-arrestin, with alternate complexes resulting in desensitization only, internalization only, or both. This project is a pilot study where Nuclear Magnetic Resonance Spectroscopy of carefully designed peptides will be used to address the following specific aims i) to determine a structural model of the C-terminus of CB1 ii) to measure the effects of phosphorylation on structure iii) to determine appropriate conditions and measure kinetics of binding of peptides to beta-arrestin. The long-term goal of this project is to attain precise knowledge of the interaction between arrestin and the receptor and it is a first step towards understanding the molecular details concerning tolerance. This knowledge will benefit human health by providing a basis for the design of ligands that selectively prevent desensitization while allowing internalization, or conversely, prevent internalization while allowing desensitization. Such ligands will be valuable as biological tools in evaluating how these processes contribute to drug addiction. In addition, this information may contribute to the design of therapeutic agents that circumvent tolerance.