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

Adenosine A receptor (AR)-dependent signaling in macrophages plays a key role in the regulation of inflammation. However, the processes regulating AR targeting to the cell surface and degradation in macrophages are incompletely understood. For example, the C-terminal domain of the AR and proteins interacting with it are known to regulate receptor recycling, although it is unclear what role potential AR-interacting partners have in macrophages. Here, we aimed to identify AR-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 AR 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 AR-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 AR is a substrate of CtsD and that the blockade of CtsD activity increases the density and cell surface targeting of AR in macrophages. Conversely, we demonstrate that AR activation prompts the maturation and enzymatic activity of CtsD in macrophages. In summary, we conclude that CtsD is a novel AR-interacting partner and thus describe molecular and functional interplay that may be crucial for adenosine-mediated macrophage regulation in inflammatory processes.