Adenosine A_2A Receptor Activation Regulates Niemann-Pick C1 Expression and Localization in Macrophages

Adenosine plays an important role in modulating immune cell function, particularly T cells and myeloid cells, such as macrophages and dendritic cells. Cell surface adenosine A_2A receptors (A_2AR) regulate the production of pro-inflammatory cytokines and chemokines, as well as the proliferation, differentiation, and migration of immune cells. In the present study, we expanded the A_2AR interactome and provided evidence for the interaction between the receptor and the Niemann-Pick type C intracellular cholesterol transporter 1 (NPC1) protein. The NPC1 protein was identified to interact with the C-terminal tail of A_2AR in RAW 264.7 and IPMФ cells by two independent and parallel proteomic approaches. The interaction between the NPC1 protein and the full-length A_2AR was further validated in HEK-293 cells that permanently express the receptor and RAW264.7 cells that endogenously express A_2AR. A_2AR activation reduces the expression of NPC1 mRNA and protein density in LPS-activated mouse IPMФ cells. Additionally, stimulation of A_2AR negatively regulates the cell surface expression of NPC1 in LPS-stimulated macrophages. Furthermore, stimulation of A_2AR also altered the density of lysosome-associated membrane protein 2 (LAMP2) and early endosome antigen 1 (EEA1), two endosomal markers associated with the NPC1 protein. Collectively, these results suggested a putative A_2AR-mediated regulation of NPC1 protein function in macrophages, potentially relevant for the Niemann-Pick type C disease when mutations in NPC1 protein result in the accumulation of cholesterol and other lipids in lysosomes.