Experimental and computational analysis of biased agonism on full-length and a C-terminally truncated adenosine A2A receptor

Biased agonism, the ability of agonists to differentially activate downstream signaling pathways by stabilizing specific receptor conformations, is a key issue for G protein-coupled receptor (GPCR) signaling. The C-terminal domain might influence this functional selectivity of GPCRs as it engages G proteins, GPCR kinases, beta-arrestins, and several other proteins. Thus, the aim of this paper is to compare the agonist-dependent selectivity for intracellular pathways in a heterologous system expressing the full-length (A(2A)R) and a C-tail truncated (A(2A)(Delta 40)R lacking the last 40 amino acids) adenosine A(2A) receptor, a GPCR that is already targeted in Parkinson's disease using a first-in-class drug. Experimental data such as ligand binding, cAMP production, beta-arrestin recruitment, ERK1/2 phosphorylation and dynamic mass redistribution assays, which correspond to different aspects of signal transduction, were measured upon the action of structurally diverse compounds (the agonists adenosine, NECA, CGS-21680, PSB-0777 and LUF-5834 and the SCH-58261 antagonist) in cells expressing A(2A)R and A(2A)(Delta 40)R. The results show that taking cAMP levels and the endogenous adenosine agonist as references, the main difference in bias was obtained with PSB-0777 and LUF-5834. The C-terminus is dispensable for both G-protein and beta-arrestin recruitment and also for MAPK activation. Unrestrained molecular dynamics simulations, at the mu s timescale, were used to understand the structural arrangements of the binding cavity, triggered by these chemically different agonists, facilitating G protein binding with different efficacy. (C) 2020 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.