PathHunter® Total GPCR Internalization cell lines are engineered to co-express the ProLink™ (PK) tagged GPCR, and an Enzyme Acceptor (EA) tag localized to the plasma membrane. Membrane bound, PK-tagged receptors are in close proximity to the EA, forcing complementation of the two β-galactosidase enzyme fragments (EA and PK). The resulting functional enzyme hydrolyzes substrate to generate a high basal chemiluminescent signal. Activation of the GPCR-PK induces internalization of the receptor, which dissociates the EA/PK complementation, and results in a loss of chemiluminescent signal.