Elucidation of G-protein and β-arrestin functional selectivity at the dopamine D2 receptor

The neuromodulator dopamine signals through the dopamine D2 receptor (D₂R) to modulate central nervous system functions through diverse signal transduction pathways. D₂R is a prominent target for drug treatments in disorders where dopamine function is aberrant, such as schizophrenia. D₂R signals through distinct G-protein and β-arrestin pathways, and drugs that are functionally selective for these pathways could have improved therapeutic potential. How D₂R signals through the two pathways is still not well defined, and efforts to elucidate these pathways have been hampered by the lack of adequate tools for assessing the contribution of each pathway independently. To address this, Evolutionary Trace was used to produce D₂R mutants with strongly biased signal transduction for either the G-protein or β-arrestin interactions. These mutants were used to resolve the role of G proteins and β-arrestins in D₂R signaling assays. The results show that D₂R interactions with the two downstream effectors are dissociable and that G-protein signaling accounts for D₂R canonical MAP kinase signaling cascade activation, whereas β-arrestin only activates elements of this cascade under certain conditions. Nevertheless, when expressed in mice in GABAergic medium spiny neurons of the striatum, the β-arrestin–biased D₂R caused a significant potentiation of amphetamine-induced locomotion, whereas the G proteinbiased D₂R had minimal effects. The mutant receptors generated here provide a molecular tool set that should enable a better definition of the individual roles of G-protein and β-arrestin signaling pathways in D₂R pharmacology, neurobiology, and associated pathologies.