KEY RESOURCES TABLE.
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| anti-Flag M1 (mouse monoclonal) | Sigma-Aldrich | Cat# F3040 RRID:AB_439712 |
| anti-Flag M2 (mouse monoclonal) | Sigma-Aldrich | Cat# F3165 RRID:AB_439685 |
| anti-HA-7 (mouse monoclonal) | Sigma-Aldrich | Cat# H9658 RRID:AB_260092 |
| anti-Erk1/2 | Cell Signaling Technology | Cat# 4695 RRID:AB_390779 |
| anti-Erk1/2 pThr202+pTyr204 | Cell Signaling Technology | Cat# 9101 RRID:AB_331646 |
| anti-Protein kinase A - catalytic subunit | Cell Signaling Technology | Cat# 5842 RRID:AB_10706172 |
| anti-Protein kinase A - catalytic subunit, pThr197 | Cell Signaling Technology | Cat# 5661 RRID:AB_10707163 |
| anti-Pan-PKA substrate pSer/pThr | Cell Signaling Technology | Cat# 9621 RRID:AB_330304 |
| anti-β2 adrenergic receptor (H-20) | Santa Cruz Biotechnology | Cat# sc-569 Lot# L0415 RRID:AB_630926 |
| anti-β2AR pSer261/pSer262 | Robert Lefkowitz (Nobles et al., 2011) | N/A |
| anti-β2AR pSer345/pSer346 | Robert Lefkowitz (Nobles et al., 2011) | N/A |
| anti-β1AR (A-20) | Santa Cruz Biotechnology | Cat# sc-567 RRID:AB_2225390 |
| anti-Phospholipase C-beta1 (D8) | Santa Cruz Biotechnology | Cat# sc-5291 RRID:AB_628118 |
| anti-Phospholipase C-beta3 (D-7) | Santa Cruz Biotechnology | Cat# sc-133231 RRID:AB_2299534 |
| anti-EpCAM (CD326) - Alexafluor488 Conjugate | BioLegend | Cat# 118210 RRID:AB_1134099 |
| anti-myeloperoxidase | Abcam | Cat# ab45977 RRID:AB_944318 |
| anti-GAPDH (6C5) | Millipore (Sigma-Aldrich) | Cat# MAB374 RRID:AB_2107445 |
| Donkey anti-rabbit-Alexafluor594 | Molecular Probes, ThermoFisher | Cat# A32754 RRID:AB_2762827 |
| Goat anti-mouse-Alexafluor568 | Molecular Probes, ThermoFisher | Cat# A11031 RRID:AB_144696 |
| Goat anti-rabbit-Alexafluor488 | Molecular Probes, ThermoFisher | Cat# A32731 RRID:AB_2633280 |
| Goat anti-rabbit-Alexafluor647 | Abcam | Cat# ab150083 RRID:AB_2714032 |
| Bacterial and Virus Strains | ||
| N/A | ||
| Biological Samples | ||
| Human heart failure patient ventricular tissue | Biorepository and Precision Pathology Center, Duke University | N/A |
| Chemicals, Peptides, and Recombinant Proteins | ||
| Thiopropyl-Sepharose 6B | Sigma-Aldrich | T8387 |
| S-Methyl methanethiosulfonate (MMTS) | Sigma-Aldrich | 64306 |
| L-NG-monomethyl arginine (L-NMMA) | Cayman Chemical | 80200 |
| L-NG-Nitroarginine methyl ester (L-NAME) | Cayman Chemical | 80210 |
| (−)-Isoproterenol (+)-bitartrate salt | Sigma-Aldrich | I2760 |
| Salbutamol | Sigma-Aldrich | S8260 |
| Salmeterol | Sigma-Aldrich | S5068 |
| Pindolol | Sigma-Aldrich | P0778 |
| Procaterol | Sigma-Aldrich | P9180 |
| Fenoterol | Sigma-Aldrich | F1016 |
| Epinephrine | Sigma-Aldrich | E4250 |
| Isoetharine | Sigma-Aldrich | I3639 |
| Pertussis toxin | Sigma-Aldrich | P7208 |
| DRAQ5 | Thermo-Fisher | 62251 |
| β-methyl cyclodextrin | Sigma-Aldrich | C4555 |
| Chlorpromazine hydrochloride | Sigma-Aldrich | C0982 |
| H-89 (N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride) | Sigma-Aldrich | B1427 |
| Genistein | Sigma-Aldrich | G6649 |
| Filipin III | Sigma-Aldrich | SAE0087 |
| Methacholine chloride | Sigma-Aldrich | 1396364 |
| 2’,5’-dideoxyadenosine 3’-triphosphate | Sigma-Aldrich | D0939 |
| KH7 ((E)-2-(1H-Benzo[d]imidazol-2-ylthio)-N’-(5-bromo-2-hydroxybenzylidene)propanehydrazide) | Sigma-Aldrich | K3394 |
| house dust mite Dermatophagoides pteronyssinus allergen | Stallergenes-Greer | B70 |
| house dust mite Dermatophagoides farinae allergen | Stallergenes-Greer | B64 |
| X-tremeGENE 9 transfection reagent | Sigma-Aldrich | 6365779001 |
| X-tremeGENE HP transfection reagent | Sigma-Aldrich | 6366244001 |
| KT-5720 | Tocris | 1288 |
| SQ-22536 | Tocris | 1435 |
| Phenylephrine hydrochloride | Sigma-Aldrich | 1533002 |
| [3H]-alprenolol; Dihydroalprenolol Hydrochloride, Levo-[Ring, Propyl-3H(N)] | PerkinElmer | NET720250UC |
| Critical Commercial Assays | ||
| PARAMETER cAMP ELISA kit | R&D Systems | KGE012 |
| GloSensor cAMP biosensor reagent | Promega | E1290 |
| Aurum Total RNA Fatty and Fibrous Tissue Kit | Bio-Rad | 7326830 |
| iScript cDNA Synthesis Kit | Bio-Rad | 1708890 |
| Fast SYBR Green Master Mix | Thermo-Fisher | 4385612 |
| TaqMan Fast Advanced Master Mix | Thermo-Fisher | 4444556 |
| cAMP Phosphodiesterase activity kit (Fluorometric) | Biovision | K2013-100 |
| Deposited Data | ||
| Image raw data files | Mendeley Data | http://dx.doi.org/10.17632/p2zycrb6dr |
| Experimental Models: Cell Lines | ||
| HEK293 | ATCC | Cat# CRL-1573 RRID:CVCL_0045 |
| HEK293 W9, stably expressing human Flag-β2AR | Robert Lefkowitz, Duke University | Shenoy et al., 2006 PMID 16280323 |
| HEK293, stably expressing HA-ATR1 | this study | N/A |
| Arrb1-/- mouse embryonic fibroblast (MEF) cell line | Robert Lefkowitz, Duke University | Kohout et al., 2001, PMID 11171997 |
| Arrb2-/- mouse embryonic fibroblast (MEF) cell line | Robert Lefkowitz, Duke University | Kohout et al., 2001, PMID 11171997 |
| Experimental Models: Organisms/Strains | ||
| Mouse: Adrb2Cys265Ser/Cys265Ser knock-in | this study | N/A |
| Mouse: Nos3 knockout: B6.129P2-Nos3tm1Unc/J | The Jackson Laboratories | Cat# Jax:002684 RRID:IMSR_JAX:002684 |
| Mouse: FLP deleter strain: B6.Cg-Tg(ACTFLPe)9205Dym/J | The Jackson Laboratories | Cat# Jax:005703 RRID:IMSR_JAX:005703 |
| Mouse: FVB.129-Adrb2tm1Bkk/J | The Jackson Laboratories | Cat# Jax: 031496 RRID:IMSR_JAX:031496 |
| Oligonucleotides | ||
| See Supplemental Table I | this study | N/A |
| Recombinant DNA | ||
| GloSensor-20F cAMP biosensor | Promega | E1171 |
| CAMYEL cAMP biosensor | Paul Sternweis, UT Southwestern, Jiang et al., 2007 PMID 17283075 | N/A |
| Flag-β2AR (human) | Robert Lefkowitz, Oppermann et al., 1996 PMID 8755530 |
N/A |
| Flag-β2AR (human), PKA− mutant | Robert Lefkowitz, Seibold et al., 1998 PMID 9516468 |
N/A |
| Flag-β2AR (human), GRK− mutant | Robert Lefkowitz, Seibold et al., 1998 PMID 9516468 |
N/A |
| GFP-β-Arrestin2 | Robert Lefkowitz, Barak et al, 1997 PMID 9346876 |
N/A |
| Venus 156-239-Gβ1, Venus 1-155-Gγ2 | Nevin Lambert, Hollins et al., 2009 PMID 19258039 |
N/A |
| β2AR-NanoLuc | Nevin Lambert, Okashah et al., 2019 PMID 31142646 |
|
| HA-ATR1 (human) | Robert Lefkowitz, Tohgo et al, 2002 PMID: 11777902 |
N/A |
| Software and Algorithms | ||
| Graphpad Prism (v7, v9) | Graphpad Software | version 7, version 9 |
| Other | ||
| N/A | ||