Cancer-Associated Mutations of the Adenosine A Receptor Have Diverse Influences on Ligand Binding and Receptor Functions.

The adenosine A receptor (AAR) is a class A G-protein-coupled receptor (GPCR). It is an immune checkpoint in the tumor micro-environment and has become an emerging target for cancer treatment. In this study, we aimed to explore the effects of cancer-patient-derived AAR mutations on ligand binding and receptor functions. The wild-type AAR and 15 mutants identified by Genomic Data Commons (GDC) in human cancers were expressed in HEK293T cells. Firstly, we found that the binding affinity for agonist NECA was decreased in six mutants but increased for the V275A mutant. Mutations A165V and A265V decreased the binding affinity for antagonist ZM241385. Secondly, we found that the potency of NECA (EC) in an impedance-based cell-morphology assay was mostly correlated with the binding affinity for the different mutants. Moreover, S132L and H278N were found to shift the AAR towards the inactive state. Importantly, we found that ZM241385 could not inhibit the activation of V275A and P285L stimulated by NECA. Taken together, the cancer-associated mutations of AAR modulated ligand binding and receptor functions. This study provides fundamental insights into the structure-activity relationship of the AAR and provides insights for AAR-related personalized treatment in cancer.