The biophysical characterization of the fundamental molecular mechanisms behind G-protein coupled receptors (GPCRs) oligomerization is proposed to be paramount for understanding the pharmacological consequence of receptor self-association. Here we developed an in vitro assay that allowed a quantitative characterization of GPCR oligomerization. The assay provided the first quantification of the association energy of the β2 Adrenergic Receptor (β2AR), a prototypical GPCR. Furthermore we directly observed the time-dependent dimerization of β2AR and Cannabinoid receptor 1 at the single molecule level, and revealed the existence of several dimerization interfaces, each with specific kinetics. Finally we investigated how a property of the
membrane solubilizing GPCRs affected oligomerization. We observed a dramatic decrease in oligomer stability with increasing geometrical membrane curvature. We anticipate that our assay will provide quantitative assessments of the functional and pharmacological consequences of GPCR
oligomerization.