Cathepsin D interacts with adenosine A2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling

Adenosine A_2A receptor (A_2AR)–dependent signaling in macrophages plays a key role in the regulation of inflammation. However, the processes regulating A_2AR targeting to the cell surface and degradation in macrophages are incompletely understood. For example, the C-terminal domain of the A_2AR and proteins interacting with it are known to regulate receptor recycling, although it is unclear what role potential A_2AR-interacting partners have in macrophages. Here, we aimed to identify A_2AR-interacting partners in macrophages that may effect receptor trafficking and activity. To this end, we performed a yeast two-hybrid screen using the C-terminal tail of A_2AR as the “bait” and a macrophage expression library as the “prey.” We found that the lysosomal protease cathepsin D (CtsD) was a robust hit. The A_2AR–CtsD interaction was validated in vitro and in cellular models, including RAW 264.7 and mouse peritoneal macrophage (IPMΦ) cells. We also demonstrated that the A_2AR is a substrate of CtsD and that the blockade of CtsD activity increases the density and cell surface targeting of A_2AR in macrophages. Conversely, we demonstrate that A_2AR activation prompts the maturation and enzymatic activity of CtsD in macrophages. In summary, we conclude that CtsD is a novel A_2AR-interacting partner and thus describe molecular and functional interplay that may be crucial for adenosine-mediated macrophage regulation in inflammatory processes.