Expression and characterization of short analogs of the Adenosine 2A Receptor

Although much is known about the structure and corresponding function of soluble proteins, the folding mechanisms and pathways of G protein‐coupled receptors (GPCRs) are poorly understood. Our goal is to understand the specific transmembrane interactions that stabilize integral membrane proteins, and to develop a model for GPCR folding. In this investigation, the Adenosine 2A Receptor (A 2A R) was studied as a model GPCR. Like all GPCRs, A 2A R contains seven membrane‐spanning alpha‐helical domains. Recombinant DNA methods were used to create a mini‐gene of the A 2A receptor that contains trans‐membrane helices two and three, and another containing five and six. Using the pET31b(+) system, fusion constructs of these peptides have been sufficiently over‐expressed in E. coli , and purified by Ni‐Chelate chromatography. Currently, studies are underway to characterize the structure of the two‐helix fragments, and measure its interactions with other trans‐membrane helices from A 2A R. Ultimately, we are using these and similar constructs to learn about the assembly pathway and structure of A 2A R. This project was funded by HHMI and the NIH.