G protein-regulated endocytic trafficking of adenylyl cyclase type 9
- Program in Biochemistry and Cell Biology, University of California San Francisco, United States
- Cardiovascular Research Institute and Department of Biochemistry and Biophysics, University of California San Francisco, United States
- Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, United States
- Lung Biology Center, Department of Medicine, University of California San Francisco, United States
- Department of Pharmacology and Moores Cancer Center, University of California San Diego, United States
- Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Japan
- Department of Psychiatry and Department of Cellular and Molecular Pharmacology, University of California San Francisco, United States
Figures
Figure 1 with 1 supplement
β2AR activation promotes redistribution of AC9 but not AC1.
(A) Representative confocal imaging of HEK293 cells coexpressing HA-β2AR and Flag-AC1 after treatment with 10 µM isoproterenol or control for 30 min. Scale Bar is 8 µm. (B) Representative confocal …
Figure 1—figure supplement 1
Functional validation of Flag-tagged AC9 and quantification of isoform-specific localization to endosomes.
(A) Quantification of the in vitro cAMP response by pcDNA3 control, untagged AC9, and Flag-tagged AC9 to 300 nM Gs (alpha subunit). (B) Quantification of the cAMP response to 10 µM isoproterenol in …
Figure 2 with 1 supplement
AC9 undergoes dynamin-dependent endocytosis and accumulates in endosomes marked by EEA1.
(A–B) Representative confocal images of HEK293 cells expressing Flag-AC1 (A) or Flag-AC9 (B) after treatment with 10 µM isoproterenol or control for 30 min and stained for endogenous EEA1. Scale bar …
Figure 2—figure supplement 1
Assessment of endosome fraction purity and wide field images of DYNGO-4a effect.
(A) Full western blot of the endosome fraction vs total cell lysate, probed for cellular markers Giantin (Golgi), Na/K pump (plasma membrane), calnexin (endoplasmic reticulum). Quantification of 3 …
Figure 3 with 1 supplement
Gs but not Gi -coupled GPCRs promote AC9 trafficking.
(A–C) Representative confocal imaging of HEK293 cells coexpressing Flag-AC9 and HA-V2R (A), HA-MOR (B), or HA-V2R-trunc (C), after treatment with 10 µM agonist (AVP or DAMGO) or control for 30 min. …
Figure 3—figure supplement 1
Validation that V2R and V2R-T activation stimulate AC9 trafficking.
(A) Representative confocal imaging of HEK293 cells expressing Flag-AC9, after treatment with 10 µM AVP or control for 30 min. Scale Bar is 8 µm. (B) Representative western blot of the surface …
Figure 4 with 3 supplements
Gs activation is sufficient to promote AC9 internalization.
(A) Representative confocal imaging of HEK293 cells cells coexpressing Flag-AC9, Gsβ, Gsγ and either HA-Gsα (HA-Gs) or HA-Gsα-CA (HA-GsCA). (B) Representative confocal imaging of HEK293 cells …
Figure 4—figure supplement 1
Confirmation that forskolin does not promote AC9 trafficking.
(A) Representative confocal imaging of HEK293 cells coexpressing HA-β2AR and Flag-AC9 were treated with 10 µM FSK or control for 30 min. Scale Bar is 5 µm. (B) Representative confocal imaging of …
Figure 4—figure supplement 2
Gs accumulation in endosomes and verification that mutational activation of Gs is sufficient to stimulate AC9 trafficking.
(A) Representative western blot of an EEA1 positive endosome fraction from HEK293 cells coexpressing Flag-AC9, Gsβ, Gsγ and HA-Gs (lane 1) or HA-GsCA (lane 2). (B) Quantification of endosome …
Figure 4—figure supplement 3
Effect of cholera toxin on AC9 trafficking.
(A) Representative confocal images of HEK293 cells coexpressing HA-β2AR and Flag-AC9 were treated with 100 nM cholera toxin overnight. Scale Bar is 8 µm. (B) Representative confocal images of HEK293 …
Figure 5 with 1 supplement
Regulation of AC9 internalization requires Gs but not beta-arrestin.
(A–B) Representative confocal imaging of Gs-knockout (GsKO) HEK293 cells coexpressing Flag-β2AR, AC9-EGFP, and either pcDNA3 (A) or wild-type HA-Gs rescue (B). (C–D) Representative confocal imaging …
Figure 5—figure supplement 1
Wide field images supporting distinct trafficking effects of Gs KO and Arr DKO.
(A) Representative wide-field images of Gs-knockout HEK293 cells coexpressing HA-β2AR and Flag-AC9 were treated with 10 µM isoproterenol for 30 min. Scale Bar is 16 µm. (B) Representative wide-field …
Figure 6 with 1 supplement
AC9 selectively contributes to the β2AR-mediated cAMP response from endosomes.
(A) Recruitment of conformational biosensors to β2AR-containing endosomes is reversed by application of the membrane permeable antagonist alprenolol for 20 min. Scale Bar is 8 µm. See Video 2 for …
Figure 6—figure supplement 1
Verification that AC9 selectively contributes to cAMP production stimulated by endogenous receptor activation in endosomes.
(A) Percent knockdown of AC9 and AC1 in HEK293 cells as determined by qPCR. [mean ± SEM; n = 3 experiments] (B) Effect of siRNA knockdown of AC9 and AC1 expression in HEK293 cells on the cAMP …
Videos
Video 1
This movie shows a confocal image series of AC9-EGFP overexpressed in a HEK293 cell.
Cell was treated with 10 µM isoproterenol added at t = 0 in the time stamp. AC9 recruitment to internal puncta is observed over the course of 30 min.
Video 2
This movie shows a confocal image series of β2AR (red) and Nb80-EGFP (green) from a HEK293 cell pre-incubated for 20 min with 100 nM isoproterenol.
10 µM alprenolol, added at t = 0 time point indicated in the time stamp, reverses Nb80-EGFP recruitment to β2ARs at both the plasma membrane and endosomes.
Video 3
This movie shows a confocal image series of β2AR (red) and Nb80-EGFP (green) from a HEK293 cell pre-incubated for 20 min with 100 nM isoproterenol.
10 µM CGP12177, added at t = 0 indicated in the time stamp, reverses Nb80-EGFP recruitment to β2ARs at the plasma membrane but not at endosomes.
Video 4
This movie shows an image series of the luminescence-based cAMP biosensor from HEK293 cells that have been treated with control siRNA (columns 1 and 2) or AC9 siRNA (columns 3 and 4).
Cells were preincubated with 100 nM isoproterenol for 20 min prior to imaging, and 10 µM CGP12177 or 10 µM alprenolol were added immediately before imaging, where indicated.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HEK293 | ATCC | CRL-1573; RRID:CVCL_0045 | Human embryonic kidney (female) |
| Cell line (Homo sapiens) | GNAS-knockout | Stallaert et al., 2017 | HEK293 parental | |
| Cell line (Homo sapiens) | Arrestin 2 and 3 double knockout | O'Hayre et al., 2017 | HEK293 parental | |
| Cell line (Homo sapiens) | ADCY 3 and 6 double knockout | Soto-Velasquez et al., 2018 | HEK293 parental | |
| Biological sample (Homo sapiens) | Human smooth airway muscle cells (HSAMs) | Prepared from lung biopsy (Tsvetanova et al., 2017) | Primary cell culture | |
| Antibody | Mouse anti-FLAG (M1) | Sigma-Aldrich | F-3040; RRID:AB_439712 | (1:1000) |
| Antibody | Rabbit anti-Flag | Sigma-Aldrich | F7425; RRID:AB_439687 | (1:1000) |
| Antibody | Mouse anti-HA | Biolegend | 16B12; RRID:AB_2820200 | (1:1000) |
| Antibody | Goat anti-AC9 | Santa Cruz Biotech | sc-8576; RRID:AB_2223286 | (1:50) |
| Antibody | Mouse anti-EEA1 | Fisher Scientific | 610457; RRID:AB_397830 | (1:1000) |
| Antibody | Mouse anti-Golgin-97 | Thermo | A-21270; RRID:AB_221447 | (1:1000) |
| Antibody | Rabbit anti-calnexin | Cell Signaling | 2679P; RRID:AB_2228381 | (1:1000) |
| Antibody | Mouse anti-Sodium/Potassium ATPase | Novus Biologicals | NB300-540SS; RRID:AB_306023 | (1:1000) |
| Recombinant DNA reagent (human) | Flag-AC9 (plasmid) | Paterson et al., 2000 | pcDNA3 backbone | |
| Recombinant DNA reagent (human) | Flag-AC1 | Chen et al., 1997 | pcDNA3 backbone | |
| Recombinant DNA reagent (human) | Flag-AC9-D442A | This study | pcDNA3 backbone | |
| Recombinant DNA reagent (human) | AC9-EGFP | This study | EGFP-C1 backbone | |
| Recombinant DNA reagent (human) | HA-B2AR | von Zastrow and Kobilka, 1992 | pcDNA3 backbone | |
| Recombinant DNA reagent (human) | HA-V2R | Rochdi et al., 2010 | pcDNA3 backbone | |
| Recombinant DNA reagent (mouse) | HA-MOR | Whistler and von Zastrow, 1998 | pcDNA3 backbone | |
| Recombinant DNA reagent (human) | HA-V2R-T | Rochdi et al., 2010 | pcDNA3 backbone | |
| Recombinant DNA reagent | Nb80-EGFP | Irannejad et al., 2013 | EGFP-C1 backbone | |
| Recombinant DNA reagent (human) | HA-G(alpha)s | Irannejad et al., 2013 | pcDNA3 backbone | |
| Recombinant DNA reagent (human) | G(beta-1) | Irannejad et al., 2013 | pcDNA3 backbone | |
| Recombinant DNA reagent (human) | G(gamma-2) | Irannejad et al., 2013 | pcDNA3 backbone | |
| Recombinant DNA reagent (human) | HA-G(alpha)s-Q227L | Masters et al., 1989 | pcDNA3 backbone | |
| Recombinant DNA reagent | pGloSensor-20F | Promega | E1171 | |
| Transfected construct (human) | AC9-siRNA | This study | See Materials and methods for sequence | |
| Transfected construct (human) | AC1 siRNA | This study | See Materials and methods for sequence | |
| Chemical compound, drug | Isoprenaline (iso) | Sigma-Aldrich | 51-30-9 | |
| Chemical compound, drug | Arginine-vasopressin (AVP) | Sigma-Aldrich | 113-79-1 | |
| Chemical compound, drug | DAMGO, [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt | Sigma-Aldrich | E7384 | |
| Chemical compound, drug | DYNGO-4a | Abcam Biochemicals | ab120689 | |
| Chemical compound, drug | Alprenolol | Sigma | 3707-88-5 | |
| Chemical compound, drug | CGP12177 | Tocris | 1134 | |
| Chemical compound, drug | Forskolin | Sigma-Aldrich | 66575-29-9 | |
| Chemical compound, drug | 3-isobutyl-1-methylxanthine (IBMX) | Sigma-Aldrich | 28822-58-4 | |
| Chemical compound | D-Luciferin, sodium salt | Gold Biosciences | LUCNA-1G | |
| Chemical compound | Anti-mouse IgG Magnetic microbeads | Miltenyi | 130-047-101 | |
| Chemical compound | EZ-link Sulfo-NHS-biotin | Pierce | 21425 | |
| Commercial assay or kit | Alexa Fluor 647 Protein Labeling Kit | Thermo Fisher Scientific | A20173 | |
| Commercial assay or kit | Alexa Fluor 488 Protein Labeling Kit | Thermo Fisher Scientific | A20181 | |
| Commercial assay or kit | Direct cAMP ELISA | Enzo Life Sciences | ADI-900–066 | |
| Software, algorithm | Prism | GraphPad | 8.1.1 | |
| Software, algorithm | ImageJ | Imagej.net | 2.0.0-rc-69/1.52 p | |
| Software, algorithm | MATLAB | MathWorks | R2014b |
