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

Abstract

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, β-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_2AR) and a C-tail truncated (A_2A ^Δ40R 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, β-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_2AR and A_2A ^Δ40R. 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 β-arrestin recruitment and also for MAPK activation. Unrestrained molecular dynamics simulations, at the μs 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.

Keywords: Adenosine A2A receptor; Functional selectivity; G protein binding; G protein coupled receptors; Molecular dynamic simulations; β-Arrestin recruitment.

Graphical abstract

Fig. 1

Competition curves in HTRF-based assays. Panel A: Scheme of the homogeneous binding technology performed in living HEK-293T cells. Panel B. Saturation isotherm of binding of fluorophore-conjugated red A_2AR ligand to HEK-293T cells transiently transfected with HALO-A_2AR in the absence (red) or presence of 10 µM SCH-58261 (black); specific binding is depicted in green. Panels C-D: Non-radiolabeled HTRF-based competition curves of specific binding of 20 nM fluorophore-conjugated red A_2AR ligand in the presence of increasing concentrations of different agonists and of the selective antagonist SCH-58261, in cells expressing A_2AR (C) or A_2A^Δ40R (D). Data represent the mean ± SEM of a representative experiment (n = 4). HTRF ratio = (665 nm acceptor signal/620 nm donor signal) × 10,000. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Ligand-receptor complexes. Cross-section through the A_2AR, highlighting the agonists adenosine (white sticks), NECA (cyan), CGS-21680 (pink), PSB-0777 (green) and LUF-5834 (blue) occupying the binding site. Color rectangles highlight regions of the orthosteric binding cavity that are occupied by functional groups of the studied agonists (see 3.2). The PIF motif (in yellow) located below the orthosteric binding cavity, and the side chain of the highly conserved R3.50 (in green) of the DRY motif near the G protein binding site are highlighted. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Signaling in cells expressing either A_2AR or A_2A^Δ40R. Dose response curves on 0.5 µM forskolin-induced cAMP levels, on ß-arrestin recruitment, on ERK1/2 phosphorylation, and on dynamic mass redistribution (DMR). Data (n = 12, each in triplicates) for cAMP are given in percentage (100% represents the forskolin effect). Data (n = 10, each in triplicates) for BRET assays, used to determine β-arrestin recruitment, are given in milliBRET Units (mBU). Data (n = 6, each in triplicates) for ERK1/2 phosphorylation are expressed as % with respect to basal levels. DMR tracings are representing the picometer (pm)-shifts of reflected light wavelengths over time upon ligand treatment.

Fig. 4

Radar plot representation of bias factors. Plots show the bias factors of the different compounds in the different functional outcomes in cells expressing A_2AR or A_2A^Δ40R. Adenosine and the Gs-cAMP signaling pathway were used as reference for calculations.

Fig. 5

Receptor side-chain movements in response to agonists. Plot of the centroids (calculated from 100 snapshots) of the C_β atoms of a selected group of 34 amino acids (Table S1) located above (in the ligand binding cavity) and below (in the G protein or β-arrestin binding cavity) the “transmission switch” amino acids obtained during 1 μs of MD simulations of A_2AR in the presence of the agonists adenosine, NECA, CGS-21680, PSB-0777 and LUF-5834 and a selective antagonist SCH-58261. The distances between the centroids of the agonist-bound conformations and the centroid of the antagonist-bound conformation were statistically correlated with E_max values measured in cAMP production and β-arrestin recruitment (Table S1). (A) Movement of the salt bridge between E169^ECL2 and H264^ECL3 that is proposed to govern the residence time of ligands. (B) Movement of E13^1.39, and the nearby H278^7.43, that correlates with β-arrestin recruitment. (C) The proposed mechanism of receptor activation at the “transmission switch” amino acids (inward movement of TM 5, an anticlockwise rotation of TM 3, and an outward movement of TM 6, see arrows) is observed but similar for full and partial agonists. (D) Movement of T88^3.36 and W246^6.48 that correlate with cAMP production (Supplementary Fig. S9).