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

Abstract

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 protein-biased 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.

Keywords: G protein; GPCR; dopamine; functional selectivity; β-arrestin.

Fig. 1.

Generation of functionally selective D₂R mutants. (A) Snake-like plot of D₂R with each round of mutagenesis color-coded according to Table S1 and Fig. S1. Red residues, derived from TYY; green spheres, predicted from piET algorithm; yellow spheres, predicted from proximity to rhodopsin/transducin Gα subunit C-terminal fragment cocrystal; gray spheres, identified residues from β2AR/Gαβγ cocrystal in intracellular loop two. Ballesteros–Weinstein numbering identified for each transmembrane domain. The long N terminus (N-term) and intracellular loop three (IC3) were abridged. The same color scheme was used to highlight the residues on the structure of D₃R (20) because D₃R is the most closely related GPCR to D₂R with an available crystal structure (81% sequence identity for transmembrane domains). (B) D₃R structure is represented as a blue ribbon, and ET-identified residues are spheres. (C) The biased mutants all occur within 20 amino acids of the DRY motif on transmembrane domain three (TM3). (D) D₃R aligned to β2AR in complex with G_αβγ (26) (green cylinder PDB ID 3SN6) as well as rhodopsin in complex with the finger-loop domain of visual arrestin (51) (purple cylinder PDB ID 4PXF). D₃R to β2AR alignment yielded an RMSD = 1.8 and D₃R alignment to rhodopsin RMSD = 2.7 using pymol MatchAlign command.

Fig. 2.

Biased D₂R mutants derived from Evolutionary Trace. (A) Inhibition of cAMP as determined by GloSensor compared with ^[WT]D₂R positive control and ^[D80A]D₂R negative control. (B) β-arrestin 2 recruitment determined by BRET for the same receptors as in A. All points are SEM of n = 3–7 done in duplicate. Confocal images of (C) ^[WT]D₂R, (D) ^[Gprot]D₂R, (E) ^[βarr]D₂R, and (F) ^[D80A]D₂R expressed in live cells. (G) B_MAX (with SEM) determined from n = 3 radioligand binding experiments. (H) K_D from B_MAX determination experiments. (I) DA competition binding experiments to determine K_I. (J) D₂R internalization assessed by live cell HA antibody staining of D₂R (SEM, n = 5 done in triplicate).

Fig. 3.

Assessment of MAP kinase activity at D₂R. (A) SRF and (B) SRE MAP kinase transcriptional promoter mediated expression of luciferase (SEM, n = 5–6 done in triplicate). (C) Western blot analysis of ERK (*P < 0.05 Newman–Keuls post hoc compared with ^[D80A]D₂R or untransfected after one-way ANOVA P < 0.05, SEM, n = 3–6) with and without β-arrestin 2 overexpression. (D) Representative blot for the data presented in C.

Fig. 4.

The physiological relevance of D₂R functional selectivity. (A) Viral transgene packaged into AAV, which allowed for Cre-dependent expression of D₂R through a double-floxed inverted ORF (DIO). (B) 0.75 µL of virus was injected bilaterally into the dorsal and ventral striatum with each injection site indicated by the red dots, and a total of 3 µL was injected into the striatum of each mouse. CPu, caudate putamen; AcbC, nucleus accumbens, core; AcbSh, nucleus accumbens, shell. (C) Representative staining pattern of the N-terminal HA tagged D₂R shows transduction of a majority of the dorsal striatum and at least 50% of the ventral striatum with variable transduction in the olfactory tubercle. Radioligand binding revealed a twofold to fourfold overexpression of each receptor as determined from membranes prepared from striatal dissections from Adora2A-Cre (D) and Adora2A-Cre::β-arrestin 2 flox (E) mice (*P < 0.05 Newman–Keuls post hoc compared with Cre (−) controls after one-way ANOVA P < 0.05, SEM, n = 4–6). (F) Potentiation of amphetamine-induced locomotion in mice when D₂R is overexpressed (*P < 0.05 bonferroni post hoc compared with ^[D80A]D₂R after repeated measures two-way ANOVA P < 0.05 for receptor expression type SEM, n = 11–12, color coded for receptor type). (G) The amphetamine response potentiation of ^[WT]D₂R and ^[βarr]D₂R is abolished when β-arrestin 2 is genetically deleted from D₂R-expressing medium spiny neurons (*P < 0.05 bonferroni post hoc compared with ^[D80A]D₂R after repeated measures two-way ANOVA P < 0.05 for receptor expression type SEM, n = 8–13).