Loss of constitutive activity is correlated with increased thermostability of the human adenosine A2A receptor

Abstract

Background and purpose: Thermostabilization by mutagenesis is one method which has facilitated the determination of high-resolution structures of the adenosine A2A receptor (A(2A)R). Sets of mutations were identified, which both thermostabilized the receptor and resulted in preferential agonist (Rag23 mutant) or antagonist (Rant5 and Rant21) binding forms as assessed by radioligand binding analysis. While the ligand-binding profiles of these mutants are known, the effects these mutations have on receptor activation and downstream signalling are less well characterized.

Experimental approach: Here we have investigated the effects of the thermostabilizing mutations on receptor activation using a yeast cell growth assay. The assay employs an engineered Saccharomyces cerevisiae, MMY24, which couples receptor activation to cell growth.

Key results: Analysis of the receptor activation profile revealed that the wild-type (WT) A(2A)R had considerable constitutive activity. In contrast, the Rag23, Rant5 and Rant21 thermostabilized mutants all exhibited no constitutive activity. While the preferentially antagonist-binding mutants Rant5 and Rant21 showed a complete lack of agonist-induced activity, the Rag23 mutant showed high levels of agonist-induced receptor activity. Further analysis using a mutant intermediate between Rag23 and WT indicated that the loss of constitutive activity observed in the agonist responsive mutants was not due to reduced G-protein coupling.

Conclusions and implications: The loss of constitutive activity may be an important feature of these thermostabilized GPCRs. In addition, the constitutively active and agonist-induced active conformations of the A(2A)R are distinct.

Figure 1

NECA-induced activity of the thermostabilized mutant A_2AR constructs (A) Rag23, Rag23-A208L and (B) Rant5 and Rant21 compared with WT. The maximum response of the WT receptor was taken as 100%, and the response obtained from the cells with no receptor was taken as 0%. The different receptor forms were expressed in the MMY24 Saccharomyces cerevisiae strain as constructs in the p306GPD vector. Data points are the average of three different experiments (two different experiments only for the Rag23-A208L mutant) performed in triplicate.

Figure 2

Saturation binding of [³H] ZM241385 and binding of the low-affinity agonist NECA in competition with [³H]ZM241385 to Saccharomyces cerevisiae MMY24 membranes expressing the WT (A), Rag23 (B), Rant5 (C) and Rant21 (D) A_2AR constructs. All data are representative of two independent experiments performed in triplicate. K_d values were derived by non-linear regression analysis of the saturation binding data. The competition binding data were fitted to a one binding site model. K_i values were derived from experimentally determined IC₅₀ values. Mean data ± SEM are presented in Table 1.

Figure 3

Functional analysis of the mutants intermediate between WT A_2AR and RANT5. The maximum response of the WT receptor was taken as 100% and the response obtained from the cells with no receptor was taken as 0%. The single mutants A54L, T88A, V239A (A) and double mutants A54L +T88A, A54L+V239A and T88A+V239A (B) are shown. The different receptor forms were expressed in the MMY24 Saccharomyces cerevisae strain as C-terminal GFP fusion constructs in the p306GPD vector. In each case, the cells were treated with increasing concentrations of the A_2AR agonist, NECA. Data points are the average of three different experiments performed in triplicate.

Figure 4

Activity of the T88A mutant compared with WT A_2AR (A, B). The maximum response of the WT receptor was taken as 100% and the response obtained from the cells with no receptor was taken as 0%. The different receptor forms were expressed in the MMY24 Saccharomyces cerevisiae strain as constructs in the p306GPD vector. The cells were treated with increasing concentrations of either NECA (A) or adenosine (B). Data points are the average of three different experiments performed in triplicate. Structures of the NECA-bound (C, Pdb accession code 2YDV) and the adenosine-bound (D, PDB accession code 2YDO) A_2AR showing the position of the T88 residue relative to the bound ligand. The T88 side chain and the ligands are shown in stick representations. The distances between the hydroxyl group of the T88 and the closest group of the two different ligands are shown by the dotted lines.

Figure 5

Superposition of helices III and VI of the inactive thermostabilized A_2AR mutant (containing the V239A mutation) structure (pink, Pdb accession code 3PWH) and the inactive A_2AR bound to an antibody (blue Pdb, accession code 3VG9). The V239 or V239A and E228 residues located on helix VI and the R102 residue located on helix III are shown as stick models. The distance between the charged interacting groups of E228 and R102 of the ionic lock in each case are indicated by the dotted lines.