Fig 1.
Arrestin residues mutated in this study.
A. Crystal structure of arrestin-3 (Protein Data Bank entry 3P2D [33]) with selected mutations indicated. Arrestin elements are colored, as follows: (Red: finger loop; Green: 157-loop; Yellow: C-loop; Blue: back-loop). B. Sequence alignment of elements containing selected mutations in arrestin-3 and other subtypes from different species. Shaded residues in each loop are the mutations selected in this study.
Fig 2.
Substitution of selected residues in arrestin-3 loops differentially affects agonist-induced binding to individual GPCRs.
BRET between Venus-tagged arrestin-3 and luciferase-tagged human M2R (A), β2AR(B), D1R(C), D2R(D) in HEK293 arrestin 2/3 KO cells. Net BRET was calculated by subtracting basal BRET (no agonist) from agonist-induced BRET. Average BRET at 10 and 15 min (means ± S.E.M.) from at least three independent experiments is shown for each arrestin-receptor pair. Each experiment was performed in quadruplicate. Statistical significance was determined using one-way ANOVA, followed by Dunnett's post-hoc test with correction for multiple comparisons. *, p< 0.05; ***, p< 0.001; ****, p<0.0001, as compared to A87V base mutant.
Fig 3.
Time course of the interaction of selected arrestins with indicated receptors.
Net BRET of selected Venus-tagged arrestin-3 mutants measured at indicated time points with luciferase-tagged M2R (A), β2AR(B), D1R(C), D2R(D) is shown (means ± S.E.M.). Note similar time dependence of A87V and ΔG65 mutant binding to the receptors, even though ΔG65 consistently demonstrates much lower binding.
Fig 4.
Deletion of G65 impedes arrestin-3 binding to D2R in vitro.
(A). Serum-starved HEK293 arrestin 2/3 KO cells were co-transfected with indicated Ve-Arr3 mutants with or without (no bait control) HA- D2R. Cells were stimulated with 10 μM quinpirole. Cleared cell lysates were immunprecipitated with HA antibody, and immunoblotted with anti-HA and anti-GFP antibodies. (B) Quantification of the specific receptor binding of different arrestin-3 mutants in three independent experiments. Non-specific binding observed in cells that do not express HA -D2R was subtracted in all cases.