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

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.

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.

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.

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.