24 research outputs found
Receptor control of partial agonism at D<sub>2</sub>R 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
Interacting partners and allosteric D<sub>2</sub>R 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: <sup>[βarr]</sup>D<sub>2</sub>R recruits normally, while <sup>[Gprot]</sup>D<sub>2</sub>R is severely deficient. (<b>C</b>) Each D<sub>2</sub>R 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<sub>αq</sub> mediated Ca<sup>2+</sup> flux, as measured by the aequorin luminescence assay, is not stimulated by <sup>[WT]</sup>D<sub>2</sub>R, <sup>[Gprot]</sup>D<sub>2</sub>R, <sup>[βarr]</sup>D<sub>2</sub>R or <sup>[D80A]</sup>D<sub>2</sub>R, compared to AngII induced Ca<sup>2+</sup> flux induced by transient expression of AT<sub>1A</sub>R. (<b>E</b>) B<sub>MAX</sub> was determined by binding, while luciferase-tagged receptors provided a B<sub>MAX</sub>-independent measure of receptor number. In this assay, the responsiveness to sodium is retained for all mutants (except <sup>[D80A]</sup>D<sub>2</sub>R). 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
Context dependent functional selectivity.
<p>(<b>A</b>) β-arrestin 2 recruitment comparing <sup>[WT]</sup>D<sub>2</sub>R and <sup>[IYIV]</sup>D<sub>2</sub>R as determined by bioluminescent resonance energy transfer (BRET). (<b>B</b>) GRK2 overexpression enhances β-arrestin 2 recruitment by BRET for <sup>[IYIV]</sup>D<sub>2</sub>R and <sup>[WT]</sup>D<sub>2</sub>R, but only slightly for <sup>[Gprot]</sup>D<sub>2</sub>R, <sup>[βarr]</sup>D<sub>2</sub>R, and <sup>[D80A]</sup>D<sub>2</sub>R 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<sub>A</sub>, calculated from EC<sub>50</sub> = 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
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 <sup>[WT]</sup>D<sub>2</sub>R for <sup>[Gprot4PM]</sup>D<sub>2</sub>R (T69F Y133L Y209N A372S). (<b>B</b>) <sup>[Gprot4PM]</sup>D<sub>2</sub>R 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 <sup>[WT]</sup>D<sub>2</sub>R 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
Agonists and antagonists with diverse pharmacophores elicit predictable responses at <sup>[Gprot]</sup>D<sub>2</sub>R and <sup>[βarr]</sup>D<sub>2</sub>R.
<p>The D<sub>2</sub>R 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, <sup>[Gprot]</sup>D<sub>2</sub>R showed a response similar to <sup>[WT]</sup>D<sub>2</sub>R at G protein activation and more similar to <sup>[D80A]</sup>D<sub>2</sub>R for β-arrestin recruitment, while <sup>[βarr]</sup>D<sub>2</sub>R 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 D<sub>2</sub>R activation at the G protein pathway (<b>G,I,K</b>) for <sup>[Gprot]</sup>D<sub>2</sub>R and <sup>[WT]</sup>D<sub>2</sub>R to the same extent, while <sup>[D80A]</sup>D<sub>2</sub>R and <sup>[βarr]</sup>D<sub>2</sub>R had no effect to inhibit. In contrast, these antagonists block DA elicited β-arrestin 2 recruitment (<b>H,J,L</b>) for <sup>[βarr]</sup>D<sub>2</sub>R and <sup>[WT]</sup>D<sub>2</sub>R. 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
GRK6 overexpression mediates βarr2 recruitment to Lgr5 intracellular vesicles.
<p>GFP-tagged βarr2 and HA epitope-tagged Lgr5 was transiently overexpressed in HEK cells (A) without GRK overexpression, or with overexpression of (B) GRK2, (C) GRK4, (D) GRK5, or (E) GRK6. Cells were fixed, permeabilized, and stained with a primary and secondary (568nm) antibody pair to HA. Representative images are presented of native GFP fluorescence (Left Panels, Green), HA-568 (Middle Panels, Red), and merged (Right Panels, yellow denotes colocalization). Arrows point to representative βarr2/Lgr5 colocalization. Inset depicts a 2x magnification. </p
Quantitation of βarr2 recruitment to Lgr5 using an ArrestinZoom Assay (Part 1).
<p>GFP-tagged βarr2 and GRK-6 were transiently co-expressed together with the permutations of Lgr5 previously described for confocal analysis and reviewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084476#pone-0084476-t001" target="_blank">Table 1</a>. (A) 48-wells of a 96-well plate were imaged for GFP fluorescence. For each well, 32 images were tiled at 108X magnification and subsequently stitched together. Stitched images were imported into ImageJ and converted to a single montage where each column represents a different experimental condition (as labeled) and each row designates a technical replicate (3.34X Magnification). As described in materials and methods, βarr2-GFP aggregates were identified (highlighted in yellow). (B) Areas outlined in (A) for (1) βarr2-GFP + GRK-6, (2) βarr2-GFP + GRK-6 + Wild-type Lgr5, and (3) βarr2-GFP + GRK-6 + S873-5A at 8.9X magnification (C) Areas outlined in B, presented at 108X magnification (Arrow in 2’ denotes βarr2 aggregates, that are absent without Lgr5 and when the “SSS” cluster is mutated to “AAA”). (D) Highlighted βarr2 aggregates in “A” were quantitated, graphed, and tested for statistically significant differences by 1-way ANOVA and <i>post </i><i>hoc</i> Bonferroni correction for multiple comparisons (p<0.05). The results are representative of three independent experiments.</p
The amino acids “SSS” from position 873-875 in Lgr5 mediate βarr2 translocation.
<p>(A) GFP-tagged βarr2 was transiently expressed alone (Left panel) or with wild-type Lgr5/-GRK6 (Middle panel) or Lgr5/+GRK6 (Right panel) in HEK 293 cells. (B) GFP-tagged βarr2 and GRK6 were transiently expressed with the indicated C-tail mutants. The primary acid sequence of the C-tail mutants is provided in tabular form (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084476#pone-0084476-t001" target="_blank">Table 1</a> 7.1-.6). (C) GFP-tagged βarr2 and Lgr4 were transiently expressed in HEK293 cells in the absence of GRK overexpression or with GRK2 or GRK6 (Left, Middle, and Right panels, respectively). Native GFP fluorescence was imaged by confocal at 100X. </p
GRK6 overexpression stimulates Class B βarr2 translocation to Lgr5 that is dependent upon a structural determinant in the C-tail.
<p>(Left panels) Wild-type HA epitope-tagged Lgr5 or (Right panels) HA epitope-tagged 834del (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084476#pone-0084476-t001" target="_blank">Table 1</a> for truncation site) Lgr5 were cotransfected in HEK293 cells with GFP-tagged βarr2 and (A) GRK2, (B) GRK4, (C) GRK5, or (D) GRK6. Cells were pulsed with an HA antibody on ice, washed, chased, and fixed. Cells were permeabilized and stained with a secondary antibody to the primary HA antibody. Images were collected on a confocal microscope at 100X. (Red:HA and Green:Native GFP were Merged:yellow). Inset depicts 2x magnification.</p
GRK overexpression mediates βarr2 translocation to Lgr5.
<p>GFP tagged βarr2 was transiently transfected in HEK 293 cells alone (Left Panels) or in the presence of overexpressed Lgr5/V2R chimera (Middle Panels) or wild-type HA epitope-tagged Lgr5 (Right Panels). βarr2-GFP translocation was visualized using a confocal microscope at 100X in (A) the absence of overexpressed GRK2 or in the presence of overexpressed (B) GRK2, (C) GRK4, (D) GRK5, or (E) GRK6. Inset depicts 2x magnification of images. </p