Abstract
Recent efforts to determine the high-resolution crystal structures for
the adenosine receptors (A1R and A2AR) 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
crystal structures obtained to date. However, the C-terminus has been
identified as a location for protein-protein interactions that may be
critical for physiological function of these important drug targets.
Here, we determine whether the presence of the full-length C-terminus
affected downstream signaling using a yeast MAPK response-based
fluorescence assay. Upon ligand binding, the A1Δ291R or A2AΔ316R
variants were unable to couple to human-yeast chimeric G-protein
chimeras to generate a downstream signal in yeast, though full-length
receptors showed native-like G-protein coupling. Further, constructs
transfected into mammalian cells (HEK-293) showed similar behavior –
i.e. the variants with C-terminal truncations lacked cAMP-linked
signaling compared to the full-length receptors. Although the C-terminus
was essential for Gα protein- associated signaling, chimeras of A1R with
a C-terminus of A2AR coupled to the A1R-specific Gα (i.e. Gαi1 versus
Gαs). This surprising result suggests that the C-terminus is important
in signaling, but not specificity, for the interaction with Gα protein.
This result has further implications in drug discovery both in enabling
the experimental use of chimeras for ligand design, and in cautious
interpretation of structure-based drug design based on truncated
receptors.