Dual mechanisms of cholesterol-GPCR interactions that depend on membrane phospholipid composition

Arka Prabha Ray, Naveen Thakur, Niloofar Gopal Pour, Matthew T. Eddy

Department of Chemistry, University of Florida, Gainesville, FL, 32611, USA

Cholesterol forms an extremely critical component of eukaryotic membrane systems and has major role in membrane organization, dynamics, membrane protein trafficking and their function^[1]. Cholesterol has been well documented to be an allosteric modulator for G protein-coupled receptors (GPCRs)^[2], sensory proteins that control numerous physiological processes and are targeted by 35% of all FDA approved drugs^[3]. Computational modelling and structural analysis have predicted the presence of putative sites for cholesterol interaction with GPCRs, but their role in preferential cholesterol interactions is an evolving field. We used ¹⁹F-NMR in aqueous solutions to decipher the structural basis of how cholesterol modulates the conformational dynamics of the human A_2A Adenosine Receptor (A_2AAR). We leverage reconstitution of A_2AAR in lipid nanodiscs to quantitatively regulate the amount of cholesterol and phospholipids in our samples over a wide range of membrane compositions. Using a covalently attached ¹⁹F-NMR probe which has previously been shown to respond to ligand efficacy^[4] and more recently to its surrounding lipid environment in nanodiscs^[5], we show that in the presence of zwitterionic lipids, A_2AAR complexes with activating drugs remained in its predominantly inactive state, while presence of cholesterol in the zwitterionic membrane environment led to activation of the receptor. Our results indicate direct receptor-cholesterol interactions drive activation of agonist-bound A_2AAR in membranes containing zwitterionic phospholipids.  Intriguingly, we observe that presence of anionic lipids attenuates cholesterol’s impact through direct interactions with the receptor, highlighting a more complex role for cholesterol that depends on membrane phospholipid composition. Furthermore, targeted amino acid replacements at two frequently predicted cholesterol interaction sites showed distinct impacts of cholesterol at different receptor locations, demonstrating the ability to delineate different roles of cholesterol in modulating receptor signaling and maintaining receptor structural integrity. Overall, our data show that cholesterol allosterically modulates the conformational dynamics of A_2AAR and provides motivation to explore the structural basis of such allosteric control of membrane protein function and systematically evaluate the potentially different roles of cholesterol at distinct receptor locations.

Acknowledgements:

This work was supported by NIH Grant R35GM138291.

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