A quantitative imaging framework reveals density-dependent GPCR oligomerization and organization in living cells
GPCR oligomerization has been reported for decades, yet its extent and functional relevance in living cells remain unresolved because existing approaches, often done in bulk, are poorly account for local receptor density, a major determinant of intermolecular interactions. Here, we establish a generic quantitative imaging framework that links spatially resolved FRET measurements describing protein oligomerization to local membrane protein in living cells. Using automated high-throughput analysis of fluorescence images, the method generates large density-resolved datasets that enable direct quantification of receptor oligomerization parameters, including apparent affinity, oligomerization state, and monomer/dimer populations at the submicrometer scale. Applied to class A GPCRs in HEK293 cells, the approach reveals receptor-specific density-dependent equilibria between monomers and dimers over physiologically relevant expression ranges, with no evidence for stable higher-order oligomers under basal conditions. The receptors studied exhibit distinct apparent affinities for dimerization, ranging from predominantly monomeric to dynamic monomer-dimer equilibria, indicating that local membrane density strongly influences receptor organization and that it is receptor dependent. The agreement between our measurements and low-density single-molecule studies further suggests that previously reported higher-order oligomers may partly reflect density-driven receptor proximity effects. By bridging single-molecule and ensemble measurements within a unified quantitative framework, this work reconciles conflicting observations in the GPCR oligomerization literature and provides a broadly applicable strategy for investigating membrane protein organization in living cells. SignificanceGPCR oligomerization in living cells is strongly influenced by the local protein density, yet most approaches do not quantitatively account for this parameter. Here, we introduce a quantitative high-throughput imaging framework that directly relates membrane protein local density to local oligomerization state in living cells. Applied to distinct GPCRs over physiologically relevant density ranges, the method reveals distinct density-dependent monomer-dimer equilibrium and apparent affinities for self-association. These results help reconcile longstanding discrepancies, where distinct oligomerization states have been measured depending on experimental conditions. More broadly, this work establishes local membrane protein density as a key determinant of membrane protein organization, and provides a quantitative framework applicable to membrane protein complexes in their native cellular context.