Engineering Chemokine Receptor Signaling Complexes In Vitro

描述（由申请人提供）：G蛋白偶联受体（gpcr）是人类最大的蛋白质超家族，有近1000个成员。这些受体通过各种刺激的转导来协调细胞间的通讯，这些刺激涉及感觉、神经传递、发育、情感、认知以及中枢神经系统、内分泌和免疫系统的功能。趋化因子受体是一类重要的gpcr，以其在免疫监视中的关键作用而闻名，它们控制白细胞的迁移和激活，以检测和解决诸如癌症和感染等生理异常。然而，这些受体的不适当表达或调节与包括炎症性疾病、癌症和艾滋病在内的大量病理有关；因此，开发阻断特定趋化因子受体功能的小分子受体拮抗剂具有重要意义。为此，我们的长期目标是获得有助于药物发现过程的结构信息。我们也有兴趣描述关于趋化因子受体结构的基本信息，与激动剂和拮抗剂诱导状态相关的动力学和构象变化，稳定受体相应的活性和非活性状态的分子相互作用，以及最终这些特性与信号输出之间的关系。然而，膜蛋白的结构、生物物理和机制信息有限。gpcr是一种特别困难的真核膜蛋白，目前只有牛视紫红质和22-肾上腺素能受体两种结构被发现。缺乏结构是由于两个主要障碍：蛋白质表达和功能重建的人工膜。然而，我们已经成功地表达了趋化因子受体CCR1，其水平足以用于生物物理研究。该项目的目的是：1)进一步优化CCR1的表达和纯化，2)优化在人工膜中制备功能性CCR1的均质制剂的条件，3)在体内表征在高亲和功能状态下生产CCR1的药理学要求，4)在体外构建涉及CCR1、趋化因子配体和异源三聚体G蛋白的三元信号复合物，5)探索趋化因子受体表达的其他异源系统。在这些研究过程中，许多重要的药理学问题将得到解决，如受体功能对G蛋白的精确要求，以及受体运输中涉及的序列基序。我们将为未来的结构和生物物理研究奠定基础，这些研究可以在一个具有良好特征的生物系统的背景下进行解释。意义：我们的研究将对趋化因子受体和gpcr的生物物理研究产生广泛的影响。鉴于大约50%的上市药物靶向gpcr，其影响可能最终扩展到趋化因子受体已成为重要治疗靶点的药物设计。公共卫生相关性：在正常生理条件下，趋化因子及其受体在免疫系统功能和发育的背景下参与以控制细胞迁移能力为中心的过程。然而，不适当的趋化因子介导的细胞迁移和炎症导致或促成许多疾病的病理，如哮喘、类风湿关节炎、多发性硬化症、心脏病和癌症。了解趋化因子的分子细节：受体的相互作用和功能，可能有助于设计治疗许多疾病的小分子和蛋白质疗法。

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) represent the largest protein superfamily in humans, with nearly 1000 members. These receptors coordinate intercellular communication via the transduction of a wide range of stimuli involved in sensation, neurotransmission, development, emotion, cognition, and function in the CNS, endocrine and immune systems. Chemokine receptors are an important class of GPCRs that are best known for their pivotal role in immune surveillance, where they control the migration and activation of leukocytes in an effort to detect and resolve physiological abnormalities such as cancer and infection. However, inappropriate expression or regulation of these receptors is associated with an extraordinary number of pathologies including inflammatory diseases, cancer and AIDS; thus there is significant interest in developing small molecule receptor antagonists that block the function of specific chemokine receptors. To this end, our long-term goal is to obtain structural information that can aid the drug discovery process. We are also interested in delineating fundamental information about chemokine receptor structures, dynamics and conformational changes associated with agonist and antagonist induced states, molecular interactions that stabilize the corresponding active and inactive states of the receptors, and ultimately the relationship between these properties and signaling output. However limited structural, biophysical and mechanistic information is available for membrane proteins. GPCRs are particularly difficult eukaryotic membrane proteins as evidenced by the fact that only two structures, bovine rhodopsin and the 22-adrenergic receptor, have been solved. The lack of structures is due to two main obstacles: protein expression and functional reconstitution in artificial membranes. However, we have succeeded in expressing the chemokine receptor, CCR1, at a level sufficient for biophysical studies. The aims of this project are to: 1) Further optimize the expression and purification of CCR1, 2) Optimize conditions for generating homogeneous preparations of functional CCR1 in artificial membranes, 3) Characterize in vivo, the pharmacological requirements for producing CCR1 in a high affinity functional state, 4) Engineer ternary signaling complexes in vitro, involving CCR1, chemokine ligands and heterotrimeric G proteins and 5) Explore alternative heterologous systems for chemokine receptor expression. In the course of these studies, many important pharmacological issues will be addressed such as the precise requirement of G proteins for receptor function, and sequence motifs involved in receptor trafficking. We will lay the foundation for future structural and biophysical studies that can be interpreted in the context of a well-characterized biological system. Significance: Our studies will have broad impact on biophysical studies of chemokine receptors and GPCRs in general. Given that approximately 50% of marketed drugs target GPCRs, impact may eventually extend to drug design where chemokine receptors have become important therapeutic targets. PUBLIC HEALTH RELEVANCE: Under normal physiological conditions, chemokines and their receptors are involved in processes centered around their ability to control cell migration in the context of immune system function and development. However, inappropriate chemokine-mediated cell migration and inflammation causes or contributes to the pathology of many diseases such as asthma, rheumatoid arthritis, multiple sclerosis, heart disease, and cancer. Understanding the molecular details of chemokine:receptor interactions and function, may facilitate the design of small molecule and protein therapeutics for many diseases.