The Specificity Of Downstream Signaling For A(1) And A(2a)R Does Not Depend On The C-Terminus, Despite The Importance Of This Domain In Downstream Signaling Strength

Recent efforts to determine the high-resolution crystal structures for the adenosine receptors (A(1)R and A(2A)R) have utilized modifications to the native receptors in order to facilitate receptor crystallization and structure determination. One common modification is a truncation of the unstructured C-terminus, which has been utilized for all the adenosine receptor crystal structures obtained to date. Ligand binding for this truncated receptor has been shown to be similar to full-length receptor for A(2A)R. However, the C-terminus has been identified as a location for protein-protein interactions that may be critical for the physiological function of these important drug targets. We show that variants with A(2A)R C-terminal truncations lacked cAMP-linked signaling compared to the full-length receptor constructs transfected into mammalian cells (HEK-293). In addition, we show that in a humanized yeast system, the absence of the full-length C-terminus affected downstream signaling using a yeast MAPK response-based fluorescence assay, though full-length receptors showed native-like G-protein coupling. To further study the G protein coupling, we used this humanized yeast platform to explore coupling to human-yeast G-protein chimeras in a cellular context. Although the C-terminus was essential for G alpha protein-associated signaling, chimeras of A(1)R with a C-terminus of A(2A)R coupled to the A(1)R-specific G alpha (i.e., G alpha i1 versus G alpha s). This surprising result suggests that the C-terminus is important in the signaling strength, but not specificity, of the G alpha protein interaction. This result has further implications in drug discovery, both in enabling the experimental use of chimeras for ligand design, and in the cautious interpretation of structure-based drug design using truncated receptors.