Background and PurposeThermostabilization by mutagenesis is one method which has facilitated the determination of high‐resolution structures of the adenosineA2Areceptor (A2AR). Sets of mutations were identified, which both thermostabilized the receptor and resulted in preferential agonist (Rag23mutant) or antagonist (Rant5andRant21) 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 ApproachHere we have investigated the effects of the thermostabilizing mutations on receptor activation using a yeast cell growth assay. The assay employs an engineeredSaccharomyces cerevisiae,MMY24, which couples receptor activation to cell growth.Key ResultsAnalysis of the receptor activation profile revealed that the wild‐type (WT)A2ARhad considerable constitutive activity. In contrast, theRag23,Rant5andRant21thermostabilized mutants all exhibited no constitutive activity. While the preferentially antagonist‐binding mutantsRant5andRant21showed a complete lack of agonist‐induced activity, theRag23mutant showed high levels of agonist‐induced receptor activity. Further analysis using a mutant intermediate betweenRag23andWTindicated that the loss of constitutive activity observed in the agonist responsive mutants was not due to reducedG‐protein coupling.Conclusions and ImplicationsThe loss of constitutive activity may be an important feature of these thermostabilizedGPCRs. In addition, the constitutively active and agonist‐induced active conformations of theA2ARare distinct.