Journal: eLife

Article Title: Distinct inactive conformations of the dopamine D2 and D3 receptors correspond to different extents of inverse agonism

doi: 10.7554/eLife.52189

Figure Lengend Snippet: In the D 2 R structure ( a ), the F-substitution on the benzisoxazole ring of risperidone occupies the Ile 3.40 sub-pocket (dotted circle) enclosed by conserved Ile 3.40 (mutated to Ala in the crystal structure to thermostabilize the receptor), Phe 5.47 , and Phe 6.44 . The same viewing angle shows that in the D 3 R ( b ) and D 4 R ( c ) structures, Cys 3.36 rotates to fill in the Ile 3.40 sub-pocket, and the substituted benzamides eticlopride and nemonapride cannot occupy the aligned sub-pockets. In our D 2 R/risperidone simulations ( d ), risperidone maintains its pose revealed by the crystal structure. In the D 2 R/spiperone simulations ( e ), the Ile 3.40 sub-pocket is similarly occupied as in D 2 R/risperidone. In the D 2 R/eticlopride simulations ( f ), the Ile 3.40 sub-pocket is collapsed as in the D 3 R ( b ) and D 4 R ( c ) structures (this trend is independent of the force field being used in the simulations).

Article Snippet: Commercial assay or kit , Spiperone-d2 SNAP-Lumi4-Tb 5x SNAP/CLIP labeling medium , Cisbio , Cat# L0002RED Cat# SSNPTBX Cat# LABMED , .

Techniques:

Journal: eLife

Article Title: Distinct inactive conformations of the dopamine D2 and D3 receptors correspond to different extents of inverse agonism

doi: 10.7554/eLife.52189

Figure Lengend Snippet: Summary of molecular dynamics simulations.

Article Snippet: Commercial assay or kit , Spiperone-d2 SNAP-Lumi4-Tb 5x SNAP/CLIP labeling medium , Cisbio , Cat# L0002RED Cat# SSNPTBX Cat# LABMED , .

Techniques:

Journal: eLife

Article Title: Distinct inactive conformations of the dopamine D2 and D3 receptors correspond to different extents of inverse agonism

doi: 10.7554/eLife.52189

Figure Lengend Snippet: Risperidone ( a, b ) and spiperone ( d, e ) similarly occupy the Ile 3.40 sub-pocket in both the presence and absence of Na + bound at the Asp80 2.50 site. In the eticlopride ( g, h ) and (-)-sulpiride ( j, k ) bound conditions, the Ile 3.40 sub-pocket is not occupied, and Cys 3.36 shows flexibility in the absence of bound Na + . ( c ), ( f ), ( i ), and l ) Distributions of the χ1 rotamer of Cys 3.36 in the D 2 R simulations in the presence of different bound ligands.

Article Snippet: Commercial assay or kit , Spiperone-d2 SNAP-Lumi4-Tb 5x SNAP/CLIP labeling medium , Cisbio , Cat# L0002RED Cat# SSNPTBX Cat# LABMED , .

Techniques:

Journal: eLife

Article Title: Distinct inactive conformations of the dopamine D2 and D3 receptors correspond to different extents of inverse agonism

doi: 10.7554/eLife.52189

Figure Lengend Snippet: The effect of mutations on the binding affinities of selected D 2 R ligands. The affinities of [ 3 H]spiperone were determined in saturation experiments at WT or mutant SNAP-tagged D 2S Rs stably expressed in FlpIn CHO cells. Binding affinity values for risperidone and eticlopride were obtained in competition binding experiments. Means of n independent experiments performed in triplicate are shown with 95% confidence intervals.

Article Snippet: Commercial assay or kit , Spiperone-d2 SNAP-Lumi4-Tb 5x SNAP/CLIP labeling medium , Cisbio , Cat# L0002RED Cat# SSNPTBX Cat# LABMED , .

Techniques: Binding Assay, Mutagenesis, Stable Transfection

Journal: eLife

Article Title: Distinct inactive conformations of the dopamine D2 and D3 receptors correspond to different extents of inverse agonism

doi: 10.7554/eLife.52189

Figure Lengend Snippet: Trp100 shows significant flexibility and can adopt multiple positions and orientations in D 2 R/eticlopride ( a–c ) and D 2 R/spiperone ( d–f ) simulations. Their χ1 and χ2 dihedral angles of Trp100 ( b, e ) and the distance between Trp100 and the ligand binding site ( c, f ) have wide and different distributions. These dihedral angle values in the D 2 R and D 3 R structures are indicated with the orange and cyan stars, respectively. The distances in the D 2 R and D 3 R structures are indicated with the orange and cyan dotted lines, respectively.

Article Snippet: Commercial assay or kit , Spiperone-d2 SNAP-Lumi4-Tb 5x SNAP/CLIP labeling medium , Cisbio , Cat# L0002RED Cat# SSNPTBX Cat# LABMED , .

Techniques: Ligand Binding Assay

Journal: eLife

Article Title: Distinct inactive conformations of the dopamine D2 and D3 receptors correspond to different extents of inverse agonism

doi: 10.7554/eLife.52189

Figure Lengend Snippet: The effect of mutations on the binding affinities of selected D 2 R ligands as determined in Tr-FRET-binding experiments. The affinities of the fluorescently labeled spiperone derivative (Spiperone-d2) or unlabeled antagonists were determined in saturation experiments at WT or mutant SNAP-tagged D 2S Rs stably expressed in FlpIn CHO cells. Binding affinity values for risperidone and eticlopride were obtained in competition binding experiments. Means of n independent experiments are shown with 95% confidence intervals (CIs).

Article Snippet: Commercial assay or kit , Spiperone-d2 SNAP-Lumi4-Tb 5x SNAP/CLIP labeling medium , Cisbio , Cat# L0002RED Cat# SSNPTBX Cat# LABMED , .

Techniques: Binding Assay, Labeling, Mutagenesis, Stable Transfection

Journal: eLife

Article Title: Distinct inactive conformations of the dopamine D2 and D3 receptors correspond to different extents of inverse agonism

doi: 10.7554/eLife.52189

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Spiperone-d2 SNAP-Lumi4-Tb 5x SNAP/CLIP labeling medium , Cisbio , Cat# L0002RED Cat# SSNPTBX Cat# LABMED , .

Techniques: Transfection, Construct, Labeling, Software