Structure-Stabilized GPCRs for Functional Analysis and Crystallography

DESCRIPTION (provided by applicant): G-Protein coupled receptors (GPCRs) are a large family of proteins expressed on the surface of eukaryotic cells. They are the major pathway for transducing extracellular signals into a diversity of intracellular responses. Because of their accessibility and physiological significance, GPCRs are the targets of approximately 30-50% of drugs on the market. GPCRs are of particular significance for mental health, since they include the receptors most neurotransmitters and are the targets of many antipsychotic and antidepressant medications, as well as drugs of abuse. However, further understanding of GPCR function is hampered by a lack of detailed structural information. The current proposal seeks to hasten the path to GPCR structure determination and structure/function studies. In Phase I we developed new tools for high-throughput cloning and membrane expression of GPCRs in E. coli. Phase II research will be centered on 10 human GPCRs implicated in brain function and mental health. We will seek to express milligram quantities of the proteins, using different transmembrane guides and indicator fusion partners. We will evaluate the proper folding of GPCRs by assaying ligand-binding activity. GPCRs that can be expressed to high levels in active form will be subject to an iterative process of solubilization, crystallization trials, and structural stabilization efforts, until suitable crystals are obtained. If necessary, we will use genetic schemes to develop modified E. coli strains for improved GPCR expression. We will also develop a system for mammalian cell expression of GPCRs that are inactive or poorly expressed in E. coli. This method will be based on cytoplasmic expression from a linear shuttle vector; importantly, the vector will be useful for bacterial expression as well. Anticipated products include novel bacterial and mammalian expression systems; bacterial and eukaryotic membrane fractions expressing GPCRs for ligand screening; GPCRs with fluorescent tags for assay development; contract expression of GPCRs for structural studies; and structural data from GPCRs for structure-based drug design. PUBLIC HEALTH RELEVANCE: G-Protein Coupled Receptors (GPCRs) are a major pathway through which cells respond to external signals, such as light, odors, hormones, or neurotransmitters. However, these proteins are difficult to express, and their detailed structure is not known. In this Phase II proposal, we plan to produce large amounts of brain-derived GPCRs to analyze their crystal structure. These studies will generate novel protein expression systems as well as structural data that can be used for basic and clinical studies of GPCRs.