Quantifying Heteromer Partitioning Reveals Inflammation-Dependent Redistribution of Microglial Adenosine A_2A and...

G protein-coupled receptor (GPCR) heteromerization represents a key organizational mechanism in cell signaling, but it remains difficult to determine, in native cells, how receptor-associated signals are distributed between non-interacting and heteromer-associated states. Here, we address this limitation by combining proximity ligation assay (PLA) with the newly applied MolBoolean methodology, enabling in situ quantification of the partitioning of adenosine A_2A and cannabinoid CB₂ receptor-associated signals between non-interacting fractions and A_2A-CB₂ heteromeric complexes in primary microglia. We show that resting microglia contain detectable A_2A-CB₂ heteromers together with a substantial non-interacting A_2A-associated signal fraction. Selective activation of either receptor promotes redistribution of the detectable receptor-associated signal toward the heteromer-associated fraction. Ligand-induced redistribution also occurred in HEK-293T cells expressing the two receptors. In contrast, pro-inflammatory activation of primary microglia with LPS/IFN-γ markedly changes the basal organization of the receptor system, increasing the proportion of MolBoolean-detectable signal associated with A_2A-CB₂ complexes, with approximately 70% of the detectable receptor-associated signal corresponding to heteromeric complexes. In this inflammatory context, further agonist-induced repartitioning is strongly limited compared with that observed in resting microglia. These findings identify inflammation-dependent receptor partitioning as a quantitatively measurable feature of microglial A_2A and CB₂ receptor organization and provide a framework for interpreting how receptor context may influence future studies of A_2A-CB₂ pharmacology under neuroinflammatory conditions.