{"doi":"10.1016/j.jbc.2022.101888","title":"Cathepsin D interacts with adenosine A2A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling","abstract":"Adenosine A2A receptor (A2AR)–dependent signaling in macrophages plays a key role in the regulation of inflammation. However, the processes regulating A2AR targeting to the cell surface and degradation in macrophages are incompletely understood. For example, the C-terminal domain of the A2AR and proteins interacting with it are known to regulate receptor recycling, although it is unclear what role potential A2AR-interacting partners have in macrophages. Here, we aimed to identify A2AR-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 A2AR 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 A2AR–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 A2AR is a substrate of CtsD and that the blockade of CtsD activity increases the density and cell surface targeting of A2AR in macrophages. Conversely, we demonstrate that A2AR activation prompts the maturation and enzymatic activity of CtsD in macrophages. In summary, we conclude that CtsD is a novel A2AR-interacting partner and thus describe molecular and functional interplay that may be crucial for adenosine-mediated macrophage regulation in inflammatory processes. Adenosine A2A receptor (A2AR)–dependent signaling in macrophages plays a key role in the regulation of inflammation. However, the processes regulating A2AR targeting to the cell surface and degradation in macrophages are incompletely understood. For example, the C-terminal domain of the A2AR and proteins interacting with it are known to regulate receptor recycling, although it is unclear what role potential A2AR-interacting partners have in macrophages. Here, we aimed to identify A2AR-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 A2AR 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 A2AR–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 A2AR is a substrate of CtsD and that the blockade of CtsD activity increases the density and cell surface targeting of A2AR in macrophages. Conversely, we demonstrate that A2AR activation prompts the maturation and enzymatic activity of CtsD in macrophages. In summary, we conclude that CtsD is a novel A2AR-interacting partner and thus describe molecular and functional interplay that may be crucial for adenosine-mediated macrophage regulation in inflammatory processes. The endogenous nucleoside adenosine plays an essential role in the regulation of homeostasis in response to stressful situations, such as low energy supply or cell damage. Extracellular adenosine is generated locally in tissues under conditions of hypoxia, ischemia, inflammation, and trauma (reviewed in Ref. (1Antonioli L. Csoka B. Fornai M. Colucci R. Kokai E. Blandizzi C. Hasko G. Adenosine and inflammation: What's new on the horizon?.Drug Discov. Today. 2014; 19: 1051-1068Crossref PubMed Scopus (110) Google Scholar)). Extracellular adenosine–mediated signaling is achieved by four G protein–coupled receptors (GPCRs), namely adenosine A1, A2A, A2B, and A3 receptors (i.e., A1R, A2AR, A2BR, and A3R, respectively) (2Fredholm B.B. AP I.J. Jacobson K.A. Klotz K.N. Linden J. International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors.Pharmacol. Rev. 2001; 53: 527-552PubMed Google Scholar). While the A1R, A2AR, and A2BR have a primary protein structure that is highly conserved during evolution, A3Rs exhibit significant sequence variations among species (3Klinger M.","journal":"Journal of Biological Chemistry","year":2022,"id":273771,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":10,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9523,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":941771,"name":"Tamás Kéki","orcid":null,"position":1,"is_corresponding":false},{"id":941293,"name":"Péter Tóth","orcid":"0000-0002-9698-8935","position":2,"is_corresponding":false},{"id":941294,"name":"Balázs Csóka","orcid":"0000-0002-7562-1130","position":3,"is_corresponding":false},{"id":941295,"name":"Balázs Koscsó","orcid":"0000-0001-9676-2066","position":4,"is_corresponding":false},{"id":464016,"name":"Zoltán H. Németh","orcid":"0000-0002-9282-6322","position":5,"is_corresponding":false},{"id":464018,"name":"Luca Antonioli","orcid":"0000-0003-1241-617X","position":6,"is_corresponding":false},{"id":266874,"name":"Andreas Ivessa","orcid":"0000-0002-7578-1337","position":7,"is_corresponding":false},{"id":290982,"name":"Francisco Ciruela","orcid":"0000-0003-0832-3739","position":8,"is_corresponding":false},{"id":941296,"name":"László Virág","orcid":"0000-0002-7099-6718","position":9,"is_corresponding":false},{"id":416900,"name":"György Haskó","orcid":null,"position":10,"is_corresponding":false},{"id":941297,"name":"Endre Kókai","orcid":"0000-0003-2035-8408","position":11,"is_corresponding":false},{"id":941770,"name":"Adrienn Skopál","orcid":null,"position":0,"is_corresponding":true}],"reference_count":64,"raw_metadata":null,"created_at":"2026-07-19T00:28:03.504676Z","pmid":"35367412","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}