Oligomerization of the Human Adenosine A2AReceptor is Driven by the Intrinsically Disordered C-Terminus

Abstract

Truncation of intrinsically disordered C-terminal segments of G protein-coupled receptors (GPCRs) facilitates the biophysical studies of these important transmembrane protein receptors, but it is unclear how such truncation affects their structure and function. The human adenosine A2A receptor (A2AR) has an unusually long (∼120 amino acid residues) disordered C-terminus that is typically truncated prior to downstream structural and functional analyses. Herein, we demonstrate that the intrinsically disordered C-terminus of A2AR drives the oligomerization of this receptor, a process that is known to modulate the function of many GPCRs. We find that A2AR homo-oligomerization decreases progressively with the shortening of the C-terminus. Mutagenesis of 355ERR357 to 355AAA357 and of C394 as well as salting-out experiments revealed that the A2AR homo-oligomer interface consists of multiple interaction sites and types, including depletion interactions involving the C-terminus. This diversity of interactions sites and types give rise to multiple possible interfaces for the A2AR oligomers. Computational modeling confirms our experimental results by showing that the C-terminus of A2AR stabilizes the homo-oligomer interface via a diversity of inter-A2AR contacts. This study uncovers the disordered C-terminus of a GPCR as the driver of oligomerization, offering important guidance for in vitro structure-function studies of A2AR and other GPCRs.