G-protein-coupled receptors (GPCR) are transmembrane proteins that play a crucial role in the communication of cells with their external environment. In the last few years, several GPCR crystal structures have been solved using genetically engineered protein. The turkey β1-adrenergic receptor, the human neutrotensin 1 receptor and the adenosine A2A receptor (A2AR) structures involved the introduction of stabilizing mutations. The engineered mutant can be stabilized in an agonist or an antagonist bound conformation making the GPCR less flexible and therefore easier to crystallize. The aim of this study was to use functional characterization of the key thermostabilising mutants of the A2AR in order to understand the molecular basis of the thermostabilisation. The different mutants were characterized using a yeast-based growth assay, which measures down-stream signaling in response to agonist and radioligand binding analysis using both an agonist and an antagonist. Point mutations leading to a reduction/loss of constitutive receptor activity have been identified. In addition, a single point mutation abolishing the ability of receptor to bind the agonist NECA has also been identified. Conformational stabilization of the receptor is thus achieved by reducing basal activity along with modifying the ligand-binding pocket leading to inability to bind agonist. Such markers can be used to screen for stable mutants for structural characterization. Since thermostabilising mutations are not directly transferable across receptors, the yeast based growth assay could serve as a quick and inexpensive way to screen for mutations for a wide range of GPCRs.