Inflammation reshapes how microglial cannabinoid receptors signal

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 A2A and cannabinoid CB2 receptor-associated signals between non-interacting fractions and A2A-CB2 heteromeric complexes in primary microglia. We show that resting microglia contain detectable A2A-CB2 heteromers together with a substantial non-interacting A2A-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 A2A-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 A2A and CB2 receptor organization and provide a framework for interpreting how receptor context may influence future studies of A2A-CB2 pharmacology under neuroinflammatory conditions.