Figure 1. Conformational biosensor detects activated D1DR at the plasma membrane and the Golgi upon dopamine (DA) stimulation.

(a) Nb6B9 binds to the receptor exclusively in its active conformation. We fused Nb6B9 to GFP and used it as a conformational biosensor for D1DR. (b) Confocal images of representative D1DR-expressing HeLa (top panel) and HEK293 cells (lower panel) with Nb6B9-GFP and GalT-mRFP expression before and after 10 µM DA addition. Stimulation with 10 µM DA results in recruitment of Nb6B9 to active D1DR at the plasma membrane and the Golgi in HeLa cells (n = 37 cells, Pearson’s coefficient = 0.62, respectively, nine biological replicates); 10 µM DA treatment only activates plasma membrane-localized D1DR in HEK293 cells (n = 17 cells, Pearson’s coefficient = 0.15, five biological replicates). Lower panels show zoomed images of insets for Snap-D1DR, Nb6BP, and the Golgi marker. Arrows indicate active D1DR at plasma membrane; arrowhead indicates active D1DR at Golgi membrane; Scale bar = 10 µm. (c) Quantification of D1DR activation at the Golgi in HeLa cells upon addition of increasing concentrations of DA; normalized fluorescence intensity of Nb6B9 at Golgi relative to Snap-tagged-D1DR at Golgi. Quantifications were baseline corrected after addition of each dose (n = 27 cells, four biological replicates). (d) Quantification of D1DR activity at Golgi in HeLa and HEK293 cells; normalized fluorescence intensity of Nb6B9 at Golgi relative to D1DR at Golgi labeled with Snap-tagged-D1DR.

Figure 1—figure supplement 1. Nb6B9 detects activated D1DR and β1AR at the plasma membrane and the Golgi in a dose-dependent manner, but not delta opioid receptors.

(a) Nanobody binding motifs are conserved between β2AR and D1DR. (b) Quantification of D1DR activation at the plasma membrane (PM) in HeLa cells upon addition of increasing dopamine (DA) concentrations; normalized fluorescence intensity of Nb6B9 at PM relative to Snap-tagged-D1DR at PM (n = 26, three biological replicates). (c) Quantification of β1AR activation at the Golgi in HeLa cells upon addition of increasing epinephrine concentrations; normalized fluorescence intensity of Nb6B9 at Golgi relative to Snap-tagged- β1AR at Golgi (n = 27, three biological replicates). (d) Quantification of β1AR activation at the PM in HeLa cells upon addition of increasing epinephrine concentrations; normalized fluorescence intensity of Nb6B9 at PM relative to Snap-tagged- β1AR at PM (n = 33, three biological replicates). Quantifications were baseline corrected after addition of each dose. (e) Quantification of delta opioid receptor (DOR) activation at the PM in HeLa cells upon addition of 10 μM D-Ala2-D-Leu2-Enkephalin (DADLE). Normalized fluorescence intensity of Nb6B9 at PM relative to GFP-DOR at the PM (n = 14, two biological replicates).

Figure 1—figure supplement 2. Conformational biosensor detects activated D1DR at the plasma membrane and the Golgi upon SKF81297 stimulation.

Representative Flag-D1DR-expressing HEK293 and HeLa cells with Nb6B9-GFP and GalT-mRFP localization at indicated times after 10 µM SKF81297 addition. Stimulation with 10 µM SKF81297 results in recruitment of Nb6B9 to active D1DR at the plasma membrane and the Golgi in both HEK293 and HeLa cells. Arrowheads indicate active D1DR at Golgi membrane; Scale bar = 10 µm.

Figure 1—figure supplement 3. MiniGαs protein biosensor detects active D1DR at the plasma membrane and the Golgi.

(a) Representative Snap-D1DR-expressing HeLa cell with miniGs-mVenus and GalT-mRFP localizations at indicated times after 10 nM SKF81297 (left panels) and dopamine (DA) (right panels) addition. Arrows indicate active D1DR at plasma membrane; Arrowheads indicate active D1DR at Golgi membrane; Scale bar = 10 µm. (b) Quantification of D1DR activation at the Golgi in HeLa cells upon addition of increasing DA concentrations; normalized fluorescence intensity of miniGs-mVenus relative to Snap-tagged-D1DR at Golgi (left) or the plasma membrane (right). Quantifications were baseline corrected after addition of each dose (n = 30, three biological replicates).

Figure 1—figure supplement 4. Conformational biosensors detect activated D1DR and Gs at the plasma membrane and the Golgi.

(a) Representative HeLa cell expressing Snap-D1DR and Nb6B9-GFP. Cells were incubated with 30 µM Dyngo-4a, a dynamin inhibitor that blocks endocytosis, stimulated with 10 µM dopamine (DA) at indicated times. Golgi-localized D1DR is activated when endocytosis is blocked, suggesting that activation of the Golgi pool is not dependent on D1DR internalization. White arrowhead indicates active D1DR at Golgi membrane; Scale bar = 10µm (b) Representative HeLa cell expressing Snap-D1DR and Nb37-GFP at indicated times after 10 µM DA addition. Nb37 binds to the GPCR-Gs protein complex in the nucleotide-free state. White arrowheads indicate active D1DR-Gs protein complex at the Golgi; yellow arrows indicate active D1DR-Gs complex at the plasma membrane; Scale bar = 10µm. (c) Quantification of Nb37-GFP and Nb6B9-GFP intensity at Golgi, normalized to Golgi-D1DR. Both nanobodies show similar kinetics of Golgi-localized D1DR activation after addition of 10 µM DA.

Figure 1—video 1. Confocal image series of D1DR-expressing HeLa cells (magenta), Nb6B9-GFP (cyan), and the Golgi marker (yellow), incubated with 10 μM dopamine.

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Figure 1—video 2. Confocal image series of D1DR-expressing HEK293 cells (magenta), Nb6B9-GFP (cyan), and the Golgi marker (yellow), incubated with 10 μM dopamine.

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Figure 1—video 3. Confocal image series of D1DR-expressing HeLa cells (magenta), Nb6B9-GFP (cyan), and the Golgi marker (yellow), incubated with 10 μM SKF81927.

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Figure 1—video 4. Confocal image series of D1DR-expressing HEK293 cells (magenta), Nb6B9-GFP (cyan), and the Golgi marker (yellow), incubated with 10 μM SKF81927.

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