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.
