Biochemical studies of retroviral receptor pseudotypes

描述(申请人提供)：膜受体是最难研究的蛋白质类别之一。逆转录病毒来源的伪型可以作为膜受体展示载体，用于基于表面等离子共振(SPR)的生物传感器研究受体与配体的相互作用。然而，这项技术的适用性取决于将膜受体整合到伪型中的能力，同时保持其结构在功能上相关的形式。这个项目的最终目标是了解可以用伪型生物传感器技术研究的受体的范围，并了解可以改变特定受体的掺入和呈现的因素。这项提议有三个具体目标。所有AIMS将广泛使用受体诱变和基于抗体的分离和检测方法。首先，我们将使用化学交联法研究异三聚体G蛋白与受体假型的结合。其次，将通过代谢标记、荧光显微镜和酶技术来研究整合到伪型中的趋化因子受体的翻译后修饰。第三，将使用化学交联法和电子显微镜检查多聚体离子通道与受体假型的结合。在所有这些研究中，将使用光学生物传感器技术来测量受体假型与其同源配体结合的能力。通过了解受体是如何结合和呈现在逆转录病毒伪型上的，我们希望拓宽可以用光学生物传感器技术研究的受体的范围。能够测量与广泛的膜受体结合的配体将有助于理解作为靶向膜受体的治疗剂的药物发现的基本生物学过程。

DESCRIPTION (provided by applicant): Membrane receptors are one of the most difficult classes of proteins to study. Retrovirus-derived pseudotypes can be used as membrane receptor display vehicles for surface plasmon resonance (SPR)-based biosensor studies of receptor-ligand interactions. However, the applicability of this technique is dependent on the ability to incorporate membrane receptors into pseudotypes while maintaining their structure in a functionally relevant form. The ultimate goal of this project is to understand the range of receptors that can be studied using pseudotype-biosensor technology and to understand the factors that can alter the incorporation and presentation of particular receptors. This proposal has three specific aims. All aims will make extensive use of receptor mutagenesis and antibody-based methods of separation and detection. First, the incorporation of heterotrimeric G proteins into receptor pseudotypes will be investigated using chemical cross-linking. Second, the post-translational modification of chemokine receptors incorporated into pseudotypes will be studied by metabolic labeling, fluorescence microscopy, and enzymatic techniques. Third, the incorporation of multimeric ion channels into receptor pseudotypes will be examined using chemical cross-linking and electron microscopy. Throughout these studies, the ability of receptor pseudotypes to bind their cognate ligand will be measured using optical biosensor technology. By understanding how receptors are incorporated and presented on retroviral pseudotypes, we hope to broaden the range of receptors that can be studies using optical biosensor technology. The ability to measure ligand binding to a wide range of membrane receptors will contribute to the understanding of basic biological processes as being a tool for drug discovery of therapeutic agents targeting membrane receptors.