Fig 1.
Context dependent functional selectivity.
(A) β-arrestin 2 recruitment comparing [WT]D2R and [IYIV]D2R as determined by bioluminescent resonance energy transfer (BRET). (B) GRK2 overexpression enhances β-arrestin 2 recruitment by BRET for [IYIV]D2R and [WT]D2R, but only slightly for [Gprot]D2R, [βarr]D2R, and [D80A]D2R when compared to [28]. All data are presented with SEM from n = 3–4 independent experiments, with statistical significance calculated in S1 Table. Quantification of bias between G protein activity (data presented in S1 Fig and[28]) and β-arrestin 2 recruitment (data presented in Fig 1A and [28]) using (C) a statistical formalism where KA, calculated from EC50 = 1 [33] or (D) bias plot mapping under normal (solid lines) and GRK2 overexpression enhanced (broken lines) conditions.
Fig 2.
Receptor control of partial agonism at D2R with A135 mutations.
(A) Relative proximity of G proteins (green spheres) and A135 (red sphere) in D3 (blue ribbon, PDB ID: 3PBL [37]) as determined by alignment of D3R to β2AR in receptor/G protein complex (PDB ID: 3SN6, [38]). (B) Arrestin (yellow spheres) does not reside close to A135 when D3R is aligned to rhodopsin in receptor/arrestin complex (PDB ID: 4ZWJ [39]). (C) G protein activity as determined by inhibition of isoproterenol-induced cAMP accumulation is titrated by substitution of A135 with a bulky polar (tyrosine) or nonpolar (phenylalanine) residue and combined with L125N or M140D to impart controlled loss of G protein function. (C) β-arrestin 2 recruitment as determined by BRET is similarly controlled. All data are presented with SEM from n = 3–5 independent experiments, with statistical significance calculated in S1 Table.
Fig 3.
A unique G protein biased mutant demonstrates agonist texture.
(A) Dopamine (DA) and quinpirole equivalently inhibit cAMP production, which is equivalent to [WT]D2R for [Gprot4PM]D2R (T69F Y133L Y209N A372S). (B) [Gprot4PM]D2R has roughly 50% efficacy in response to DA but not quinpirole for β-arrestin 2 recruitment. (C) GRK2 overexpression rescues both DA and quinpirole β-arrestin 2 recruitment activity nearly to [WT]D2R levels (dotted line, from Fig 1B). (D) GRK2 recruitment as determined by BRET (where GRK2 is tagged with YFP) shows the same ligand discrepancy as β-arrestin 2. All data are presented with SEM from n = 3 independent experiments, with statistical significance calculated in S3 Table.
Fig 4.
Agonists and antagonists with diverse pharmacophores elicit predictable responses at [Gprot]D2R and [βarr]D2R.
The D2R agonists quinpirole, apomorphine, and N-propylapomorphine (NPA) were tested for G protein activity (A,C,E) and β-arrestin 2 recruitment (B,D,F). For each agonist, [Gprot]D2R showed a response similar to [WT]D2R at G protein activation and more similar to [D80A]D2R for β-arrestin recruitment, while [βarr]D2R was not active at the G protein pathway but retained activity at the β-arrestin pathway. The antagonists raclopride (G,H) haloperidol (I,J) and partial antagonist aripiprazole (K,L) were able to block DA elicited D2R activation at the G protein pathway (G,I,K) for [Gprot]D2R and [WT]D2R to the same extent, while [D80A]D2R and [βarr]D2R had no effect to inhibit. In contrast, these antagonists block DA elicited β-arrestin 2 recruitment (H,J,L) for [βarr]D2R and [WT]D2R. All data are presented with SEM from n = 3–4 independent experiments, with statistical significance calculated in S3 Table.
Fig 5.
Interacting partners and allosteric D2R determinants of functional selectivity.
(A) GRK2 and (B) β-arrestin 1 recruitment as assessed by BRET show a similar profile as β-arrestin 2: [βarr]D2R recruits normally, while [Gprot]D2R is severely deficient. (C) Each D2R construct was expressed in HEK 293T cells and assessed for its ability to stimulate cAMP in response to DA. Stimulation of endogenous receptor by isoproterenol was used as a control response. (D) Gαq mediated Ca2+ flux, as measured by the aequorin luminescence assay, is not stimulated by [WT]D2R, [Gprot]D2R, [βarr]D2R or [D80A]D2R, compared to AngII induced Ca2+ flux induced by transient expression of AT1AR. (E) BMAX was determined by binding, while luciferase-tagged receptors provided a BMAX-independent measure of receptor number. In this assay, the responsiveness to sodium is retained for all mutants (except [D80A]D2R). All data are presented with SEM from n = 3–4 independent experiments, with statistical significance calculated in S4 Table.