The organic cation transporter 2 regulates dopamine D1 receptor signaling at the Golgi apparatus
- Department of Biochemistry & Biophysics, University of California, San Francisco, United States
- Biochemistry Department, Weill Cornell Medicine, United States
- Cardiovascular Research Institute, University of California, San Francisco, United States
Figures
Figure 1 with 8 supplements
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 …
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) …
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 …
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. …
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 …
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.
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.
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.
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.
Figure 2 with 5 supplements
OCT2 facilitated dopamine (DA) transport to the Golgi-localized D1DR.
(a) Representative HeLa cell expressing Snap-D1DR, Nb6B9-GFP, and GalT-mRFP expression pretreated with 100 μM imipramine for 15 min, before and after 10 μM DA addition. Inhibition of OCT2 blocks …
Figure 2—figure supplement 1
OCT2 expression is required for facilitated transport of DA to the Golgi-localized D1DR.
(a) Dose–response curve of forskolin-normalized D1DR-mediated cAMP response in HEK293 cells treated with SKF81297 or dopamine (DA); SKF and DA have similar potency and efficacy, thus differences in …
Figure 2—figure supplement 2
OCT2, but not OCT3, is required for DA-mediated D1DR activation at the Golgi.
(a) Representative HeLa cell expressing Snap-D1DR, Nb6B9-GFP, and GalT-mRFP pretreated with 10 μM corticosterone, an OCT3-selective inhibitor, for 15 min, before and after 10 µM dopamine addition. …
Figure 2—video 1
Confocal image series of D1DR-expressing HeLa cells (magenta), Nb6B9-GFP (cyan), and Galt-mApple (yellow), pretreated with 100 μM imipramine and incubated with 10 μM dopamine and SKF81297.
Figure 2—video 2
Confocal image series of D1DR-expressing HEK293 cells (magenta), Nb6B9-GFP (cyan), and OCT2-mApple (yellow), incubated with 10 μM dopamine.
Figure 2—video 3
Confocal image series of D1DR-expressing HeLa cells (magenta), miniGs-mApple (yellow), expressing SLC22A2 shRNA-B and incubated with 10 μM Dopamine for 20 min and then 10 μM SKF81297.
Figure 3 with 3 supplements
Regulation of dopamine (DA)-mediated activation of the Golgi-localized D1DR in striatal neurons by OCT2.
(a) Representative medium spiny neurons (MSN) expressing Snap-D1DR and Nb6B9-GFP at indicated times after 10 µM DA addition. DA stimulates D1DR activation at the Golgi in MSNs (n = 22 cells, …
Figure 3—figure supplement 1
Medium spiny neurons endogenously express OCT2 and D1DR at the plasma membrane and the Golgi.
(a) Detection of OCT2 expression in different tissue extracts and brain slices by Western blot. (b) Top panel: colocalization of Nb6B9-GFP at the Golgi after 10 μM dopamine (DA) stimulation for 10 …
Figure 3—video 1
Confocal image series of D1DR-expressing medium spiny neurons (MSNs) (magenta) and Nb6B9-GFP (cyan), incubated with 10 μM dopamine.
Figure 3—video 2
Confocal image series of D1DR-expressing medium spiny neurons (MSNs) (magenta) and Nb6B9-GFP (cyan), pretreated with 100 μM imipramine and incubated with 10 μM dopamine and SKF81297.
Figure 4
Both plasma membrane and Golgi-localized D1DR promote cAMP production.
Model of blocking D1DR-Gs coupling at the plasma membrane (PM) (a) and the Golgi membrane (b) after recruitment of mVenus-FRB-Nb6B9. FKBP was targeted to either the PM (a) or the Golgi membrane (b), …
Figure 5 with 1 supplement
Golgi-localized protein kinase A (PKA) is activated by D1DR at the Golgi.
(a) Model of PKA activation; cAMP binds PKA regulatory subunit (R), rendering PKA catalytic subunit (PKA-cat) dissociation. (b) Confocal images of representative D1DR-expressing HEK293 cells with …
Figure 5—figure supplement 1
Kinetics of PKAcat-GFP dissociation from the Golgi membrane upon activation of the D1DR Golgi pools.
Figure 6 with 1 supplement
Rapid activation of Golgi-localized D1DR and protein kinase A (PKA) by photo-release of dopamine.
(a) Dopamine is uncaged from (N)–1-(2 nitrophenyl) ethylcarboxy-3,4-dihydroxyphenethylamine (NPEC) upon blue light (UV) exposure. (b) Confocal images of representative D1DR-expressing HEK293 cells …
Figure 6—video 1
Confocal image series of D1DR-expressing (magenta) HEK293 cells with endogenous PKA-cat-GFP (cyan) and Nb6B9-mApple (orange), incubated with 1 μM (N)–1-(2 nitrophenyl) ethylcarboxy-3,4-dihydroxyphenethylamine (NPEC)-caged dopamine.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information | |
|---|---|---|---|---|---|
| Cell line (Homo sapiens) | HEK293 | ATCC | CRL-1573.3 | Mycoplasma tested negative | |
| Cell line (H. sapiens) | HEK293T | ATCC | ACS-4500 | Mycoplasma tested negative | |
| Cell line (H. sapiens) | HeLa | ATCC | CRM-CCL-2 | Mycoplasma tested negative | |
| Antibody | Anti-dopamine receptor D1(rabbit polyclonal) | Abcam | ab216644 | IF (1:100) | |
| Antibody | Anti-GM130 (mouse monoclonal) | BD Biosciences | 610822 | IF (1:1000) | |
| Antibody | Anti-SLC22A2 (rabbit polyclonal) | Abcam | ab170871 | WB (1:1000) | |
| Antibody | Anti-SLC22A2 (rabbit polyclonal) | ABclonal | A14061 | IF (1:100) | |
| Antibody | Anti-GAPDH (mouse monoclonal) | Proteintech | 60004-1 | WB (1:10,000) | |
| Antibody | Anti-HRP-conjugated IgG (rabbit polyclonal) | GE Healthcare | P132460 | WB (1:10000) | |
| Antibody | Anti-mouse IgG (donkey polyclonal) | Thermo Fisher | A32766 | IF (1:10,000) | |
| Antibody | Anti-rabbit IgG (donkey polyclonal) | Thermo Fisher | A32794 | IF (1:10,000) | |
| Antibody | Anti-dopamine receptor D1 (rabbit polyclonal) | Proteintech | 17934-1-AP | IF (1:100) | |
| Peptide, recombinant protein | D1DR blocking peptide | Proteintech | Ag12366 | IF (1:25) | |
| Chemical compound, drug | Snap-Cell 647 | NEB | S9102S | ||
| Chemical compound, drug | Dopamine hydrochloride | Sigma | 200-527-8 | ||
| Chemical compound, drug | SKF81297 hydrobromide | Tocris | 1447 | ||
| Chemical compound, drug | NPEC-caged-dopamine | Tocris | 3992 | ||
| Chemical compound, drug | A/C heterodimerizer | Takara | 635056 | ||
| Chemical compound, drug | Dyngo | Abcam | ab120689 | ||
| Chemical compound, drug | Forskolin | Sigma | F6886-10MG | ||
| Chemical compound, drug | Imipramine | Sigma | 113-52-0 | ||
| Chemical compound, drug | Corticosterone | Sigma | 200-019-6 | ||
| Software, algorithm | Prism | GraphPad | |||
| Software, algorithm | ImageJ | Imagej.net/contributors | |||
| Software, algorithm | MATLAB R2014b | MathWorks | DOI:10.5281/zenodo.5146169 | Bakr et al., 2021 | |
| Recombinant DNA reagent | pGloSensor-20F | Promega | E1171 | ||
| Recombinant DNA reagent | Signal sequence Snap-D1DR | This study | pCDNA3 backbone; snap vector, see Materials and methods | ||
| Recombinant DNA reagent | pVenus-FRB-Nb6B9 | This study | pVenus-C1 vector, see Materials and methods | ||
| Recombinant DNA reagent | FKBP-GalT-mApple | This study | pm-Apple-M1 vector, see Materials and methods | ||
| Recombinant DNA reagent | Lyn-2xFKBP-CFP | Addgene | 20149 | ||
| Recombinant DNA reagent | pCAG-Snap-D1DR | This study | pCAG vector, see Materials and methods | ||
| Recombinant DNA reagent | pCAG-Nb6B9-GFP pCAG-Nb37-GFP | This study | pCAG vector, see Materials and methods | ||
| Sequence-based reagent | SLC22A2 shRNAs | OriGene | TL517269 | pGFP-C-shLenti | |
| Sequence-based reagent | Scamble shRNAs | OriGene | TR30021 | pGFP-C-shLenti | |
| Biological sample (Mus musculus, male and female) | CD1 | Charles Rivers | Crl:CD1(ICR) | Isolated medium spiny neurons from neonatal mouse striatal, see Material and methods | |
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/75468/elife-75468-transrepform1-v2.pdf
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Source data 1
Source data of all figures and figure supplements are combined in one file, separated and labeled accordingly in each tab.
- https://cdn.elifesciences.org/articles/75468/elife-75468-data1-v2.xlsx
