(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 …
(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) …
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 …
(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. …
(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 …
(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 …
(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 …
(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. …
(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, …
(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 …
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), …
(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 …
(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 …
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 |
Source data of all figures and figure supplements are combined in one file, separated and labeled accordingly in each tab.