Receptor control of partial agonism at D₂R with A135 mutations.

<p>(<b>A</b>) Relative proximity of G proteins (green spheres) and A135 (red sphere) in D3 (blue ribbon, PDB ID: 3PBL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141637#pone.0141637.ref037" target="_blank">37</a>]) as determined by alignment of D3R to β2AR in receptor/G protein complex (PDB ID: 3SN6, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141637#pone.0141637.ref038" target="_blank">38</a>]). (<b>B</b>) Arrestin (yellow spheres) does not reside close to A135 when D3R is aligned to rhodopsin in receptor/arrestin complex (PDB ID: 4ZWJ [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141637#pone.0141637.ref039" target="_blank">39</a>]). (<b>C</b>) 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. (<b>C</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141637#pone.0141637.s003" target="_blank">S1 Table</a>.</p

Context dependent functional selectivity.

<p>(<b>A</b>) β-arrestin 2 recruitment comparing ^[WT]D₂R and ^[IYIV]D₂R as determined by bioluminescent resonance energy transfer (BRET). (<b>B</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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141637#pone.0141637.ref028" target="_blank">28</a>]. All data are presented with SEM from n = 3–4 independent experiments, with statistical significance calculated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141637#pone.0141637.s003" target="_blank">S1 Table</a>. Quantification of bias between G protein activity (data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141637#pone.0141637.s001" target="_blank">S1 Fig</a> and[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141637#pone.0141637.ref028" target="_blank">28</a>]) and β-arrestin 2 recruitment (data presented in Fig 1A and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141637#pone.0141637.ref028" target="_blank">28</a>]) using (<b>C</b>) a statistical formalism where K_A, calculated from EC₅₀ = 1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141637#pone.0141637.ref033" target="_blank">33</a>] or (<b>D</b>) bias plot mapping under normal (solid lines) and GRK2 overexpression enhanced (broken lines) conditions.</p

Agonists and antagonists with diverse pharmacophores elicit predictable responses at ^[Gprot]D₂R and ^[βarr]D₂R.

<p>The D₂R agonists quinpirole, apomorphine, and N-propylapomorphine (NPA) were tested for G protein activity (<b>A,C,E</b>) and β-arrestin 2 recruitment (<b>B,D,F</b>). 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 (<b>G,H</b>) haloperidol (<b>I,J</b>) and partial antagonist aripiprazole (<b>K,L</b>) were able to block DA elicited D₂R activation at the G protein pathway (<b>G,I,K</b>) 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 (<b>H,J,L</b>) for ^[βarr]D₂R and ^[WT]D₂R. All data are presented with SEM from n = 3–4 independent experiments, with statistical significance calculated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141637#pone.0141637.s005" target="_blank">S3 Table</a>.</p

Interacting partners and allosteric D₂R determinants of functional selectivity.

<p>(<b>A</b>) GRK2 and (<b>B</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. (<b>C</b>) 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. (<b>D</b>) G_αq 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 AT_1AR. (<b>E</b>) B_MAX was determined by binding, while luciferase-tagged receptors provided a B_MAX-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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141637#pone.0141637.s006" target="_blank">S4 Table</a>.</p

A unique G protein biased mutant demonstrates agonist texture.

<p>(<b>A</b>) Dopamine (DA) and quinpirole equivalently inhibit cAMP production, which is equivalent to ^[WT]D₂R for ^[Gprot4PM]D₂R (T69F Y133L Y209N A372S). (<b>B</b>) ^[Gprot4PM]D₂R has roughly 50% efficacy in response to DA but not quinpirole for β-arrestin 2 recruitment. (<b>C</b>) GRK2 overexpression rescues both DA and quinpirole β-arrestin 2 recruitment activity nearly to ^[WT]D₂R levels (dotted line, from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141637#pone.0141637.g001" target="_blank">Fig 1B</a>). (<b>D</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141637#pone.0141637.s005" target="_blank">S3 Table</a>.</p