CBE Seminar (Zoom): G-Protein Coupled Receptors -- Protein Engineering to Improve Expression but Retain Function

Registration Link: https://forms.gle/NvYfQ34LCDBuDdJr6

Abstract: The adenosine receptor subfamily are membrane-localized G-protein coupled receptors that modulate blood pressure and most recently have been implicated in cancer, neurodegenerative diseases and diabetes, making them a significant fraction of drug discovery efforts. Adenosine A_2A receptor (A_2AR), one of four subfamily members, has a longer C-terminus than the other adenosine receptor subtypes, which may contribute to its exceptional trafficking to the plasma membrane, but its flexibility has been attributed to enabling several important protein-protein interactions. In contrast to A_2AR, related family members A₁ and A₃ are poorly expressed in all expression hosts. We created chimeric A₁ and A₃ receptors with A_2AR C-termini that show improved localization to the plasma membrane, with greater than three-fold higher yields than previously reported from other heterologous expression systems. Interestingly, these chimeras are able to bind to their selective ligands as well as native G proteins, suggesting the specificity of this interaction does not rely on the C-terminus. To investigate the importance of the C-termini further, we purified A_2AR and its cognate Gαs to determine the affinity and binding rates via surface plasmon resonance (SPR). We find that a commonly used truncation for crystallography – A_2A316R– was capable of binding Gαs, but with a significantly decreased total complex formation, as well as an increased dissociation rate compared to full length receptor. Together with in vivo signaling data, our results suggest that loss of downstream signaling for the truncation may result from reduced GPCR-G protein complex formation, which may be important for drug discovery based on the crystal structures alone.

Bio: Anne Skaja Robinson became head of Carnegie Mellon University’s Department of Chemical Engineering in 2018 and Trustee Professor of chemical engineering in 2019. Prior to her current appointment, she served as chair of the Department of Chemical and Biomolecular Engineering and Boh Professor of engineering at Tulane University. She started her academic career at the University of Delaware, where she ultimately became a full professor and associate chair in chemical engineering. Having received both bachelor's and master's degrees in chemical engineering from Johns Hopkins University, and her doctorate in chemical engineering from the University of Illinois at Urbana-Champaign, Robinson has earned many honors, including a DuPont Young Professor Award and a National Science Foundation Presidential Early Career Award for Science and Engineering. She is also a fellow of both the American Institute for Medical and Biological Engineering and the American Institute of Chemical Engineers. Robinson’s research focuses on three primary areas of bioengineering: expression and characterization of integral membrane proteins, especially G-protein coupled receptors; understanding and controlling protein aggregation; and cellular mechanisms controlling protein quality and human disease.