KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Bacterial and virus strains | ||
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| Escherichia coli BL21(DE3) | Invitrogen | Cat# C600003 |
| MAX Efficiency DH10Bac Competent Cells | Thermo Fisher Scientific | Cat# 10,361,012 |
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| Chemicals, peptides, and recombinant proteins | ||
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| GDP | Sigma-Aldrich | Cat# G7127–100MG |
| GTP | Sigma-Aldrich | Cat# 11,140,957,001 |
| ATP | Discoverx | Cat# 90–0099 |
| Guanosine 5’-[β,γ-imido]triphosphate (GNP, GppNHp) | Axorra | Cat# JBS-NU-401–50 |
| 100X GTPγS, 10mM | EMD Millipore | Cat# 20–176 |
| Guanosine 5ʹ-Diphosphate, Trisodium Salt, [8,50–3H]-, Specific Activity: 25–50Ci (0.925–1.85TBq)/mMole, 250 μCi (9.25MBq) | Perkin-Elmer | Cat# NET966250UC |
| GTP, [γ−32P]-6000 Ci/mmol 10 mCi/ml Lead, 250 μCi | Perkin-Elmer | Cat# NEG004Z250UC |
| GTPγS, [35S]-1250 Ci/mmol, 12.5 mCi/ml, 250 μCi | Perkin-Elmer | Cat# NEG030H250UC |
| Forskolin | Cayman Chemical Company | Cat# 11,018: 50 mg |
| Isoproterenol Hydrochloride | TCI | Cat# I0260 |
| Acetylcholine Chloride | Selleckchem | Cat# S1805 |
| Activated Charcoal Norit | Sigma-Aldrich | Cat# 53,663–250G |
| Cytoscint-ES liquid scintillation cocktail | MP Biomedicals | Cat# 0,188,245,301 |
| Acrylonitrile | Sigma-Aldrich | Cat# 110,213–5ML |
| cOmplete Protease Inhibitor Cocktail | Sigma-Aldrich | Cat# 5,056,489,001 |
| Carbenicillin | Goldbio | Cat# C-103–100 |
| Kanamycin | Goldbio | Cat# K-120–25 |
| IPTG | Goldbio | Cat# I2481C100 |
| DTT | Goldbio | Cat# DTT10 |
| Biotin | Sigma-Aldrich | Cat# B4501–5G |
| M-MLV reverse transcriptase | Promega | Cat# 3683 |
| acetylated BSA | Nacalai Tesque | Cat# 01,278–44 |
| TrypLE™ Express Enzyme (1X), no phenol red | Fisher Scientific | Cat# 12,604,013 |
| PBS, pH 7.4 | Thermo Fisher Scientific | Cat# 10,010,049 |
| 3-Isobutyl-1-methylxanthine, BioUltra, ≥99% (IBMX) | Sigma-Aldrich | Cat# I7018 |
| DMSO sterile filtered | Sigma-Aldrich | Cat# D2650 |
| ct-TAMRA | Promega | Cat# G8251 |
| Coelenterazine-400a (Nanolight Technology) | Prolume Ltd | Cat# 340–1 |
| BSA, Fraction V, low Heavy Metals | EMD Millipore | Cat# 12,659–100GM |
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| Critical commercial assays | ||
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| LANCE Ultra cAMP Detection Kit | Perkin-Elmer | Cat# TRF0263 |
| GTPase Colorimetric Assay Kit 480 Tests | Innova Biosciences | Cat# 602–0121 |
| Pierce™ BCA® Protein Assay Kits and Reagents, Thermo Scientific, BCA | Fisher Scientific | Cat# PI23227 |
| Streptavidin XL665 | Cisbio | Cat# 610SAXLF |
| Anti-6His-Tb cryptate | Cisbio | Cat# 61HI2TLF |
| Green Up cADDis cAMP Assay Kit | Montana Molecular | Cat# U0200G |
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| Deposited data | ||
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| GppNHp-bound Gαs in complex with the cyclic peptide inhibitor GN13 | This paper | PDB: 7BPH |
| GDP-bound Gαs in complex with the cyclic peptide inhibitor GD20 | This paper | PDB: 7E5E |
| GTPγS-bound Gαs | Sunahara et al. (1997) | PDB: 1AZT |
| GTPγS-bound Gαs in complex with adenylyl cyclase | Tesmer et al. (1997) | PDB: 1AZS |
| GDP-bound Gαs in complex with Gβγ | Liu et al. (2019) | PDB: 6EG8 |
| GDP•AlF4--bound Gα13 in complex with PRG rgRGS domain | Chen et al. (2008) | PDB: 3CX7 |
| GDP•AlF4--bound Gαq in complex with RGS8 | Taylor et al. (2016) | PDB: 5DO9 |
| GDP•AlF4--bound Gαi in complex with KB1573 | Johnston et al. (2006) | PDB: 2G83 |
| GDP-bound Gα13 in complex with PRG rgRGS domain | Chen et al. (2008) | PDB: 3CX6 |
| GDP-bound Gαq in complex with Gβγ and YM-254890 | Nishimura et al. (2010) | PDB: 3AH8 |
| GDP-bound Gαi in complex with Gβγ | Wall et al. (1995) | PDB: 1GP2 |
| GDP•AlF4--bound Gα(t/i) in complex with RGS9 and PDEγ | Slep et al. (2001) | PDB: 1FQJ |
| Gαs/Gβγ/β2AR/Nb35 complex | Rasmussen et al. (2011) | PDB: 3SN6 |
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| Experimental models: Cell lines | ||
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| Sf9 cells | Thermo Fisher Scientific | Cat# 12,659,017 |
| Halo-Tag-GFP-Mito expressing HeLa cells | J. Kritzer (Tufts University) (Peraro et al., 2018) | N/A |
| HEK293 cells | ATCC | Cat# CRL-1573 |
| (Parent) HEK293 cells | A. Inoue (Tohoku University) (Stallaert et al., 2017) | N/A |
| GNAS KO HEK293 cells (CL4) | A. Inoue (Tohoku University) (Stallaert et al., 2017) | N/A |
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| Recombinant DNA | ||
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| Gαs(WT) cloned into a modified pET15b vector | (Hu and Shokat, 2018) | N/A |
| Gαs(Q227L) cloned into a modified pET15b vector | This study | N/A |
| Human ADCY2 (residues 871–1082) cloned into a modified pET15b vector | (Hu and Shokat, 2018) | N/A |
| Mouse ADCY5(D628E/S645R) (residues 443–659) cloned into a pET29b vector | (Hu and Shokat, 2018) | N/A |
| HumanGNB1(WT) and GNG2(C68S) cloned into a modified pFastBac Dual vector | (Hu and Shokat, 2018) | N/A |
| Avi-Gαs(WT) cloned into a modified pET15b vector | This study | N/A |
| Avi-Gαi1(WT) cloned into a modified pET15b vector | This study | N/A |
| Avi-Gαs(S275L) cloned into a modified pET15b vector | This study | N/A |
| pcDNA3 Gαs(WT)-HA | This study | N/A |
| pcDNA3 Gαs(S275L)-HA | This study | N/A |
| pcDNA3 Gαsi1(WT)-EE-tagged | G. Peng (UCSF) | N/A |
| SSF-β2AR | B. Barsi-Rhyne (UCSF) | N/A |
| pCEH-Sero-SNAP-hM2R | R. Mackinnon (The Rockefeller University) | N/A |
| Gβ1-C Venus | R. Mackinnon (The Rockefeller University) | N/A |
| Gγ2-N Venus | R. Mackinnon (The Rockefeller University) | N/A |
| GIRK4-NLuc | R. Mackinnon (The Rockefeller University) | N/A |
| pcDNA3.1-Beta1 | Olsen et al. (2020) | Addgene plasmid # 140,987 |
| pcDNA3.1-GGamma1-GFP2 | Olsen et al. (2020) | Addgene plasmid # 140,989 |
| pcDNA3.1-GGamma2-GFP2 | This paper | N/A |
| pcDNA5/FRT/TO-GAlphai1-RLuc8 | Olsen et al. (2020) | Addgene plasmid # 140,973 |
| pcDNA5/FRT/TO-GAlphasS-RLuc8 | Olsen et al. (2020) | Addgene plasmid # 140,980 |
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| Software and algorithms | ||
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| GraphPad Prism | GraphPad Software | https://www.graphpad.com/scientific-software/prism/ |
| CCP4 | Winn et al. (2011) | http://www.ccp4.ac.uk/ |
| Phenix | Adams et al. (2010) | https://www.phenix-online.org/ |
| Coot | Emsley et al. (2010) | https://www2.mrc-lmb.cam.ac.uk/%20personal/pemsley/coot/ |
| Excel | Microsoft | https://www.microsoft.com/en-us/ |
| Word | Microsoft | https://www.microsoft.com/en-us/ |
| Illustrator | Adobe | https://www.adobe.com/products/illustrator.html |
| Pymol | The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC. | https://pymol.org/2/ |
| UCSF Chimera | Pettersen et al. (2004) | https://www.cgl.ucsf.edu/chimera |
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| Other | ||
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| TALON Metal Affinity Resin | Clontech Laboratories | Cat# 635,503 |
| SOURCE 15Q, 200 mL | GE Healthcare | Cat# 17–0947-05 |
| Superdex 200 Increase 10/300 GL | GE Healthcare | Cat# 28–9909-44 |
| Sephadex G-25 | GE Healthcare | Cat# 17,003,201 |
| Dynabeads M280 streptavidin magnetic beads | Thermo Fisher Scientific | Cat# 11206D |
| Transit 2020 | Fisher Scientific | Cat# MIR5404 |
| Lipofectamine 2000 Transfection Reagent | Thermo Fisher Scientific | Cat# 11,668,019 |
| Opti-MEM™ I Reduced Serum Medium | Fisher Scientific | Cat# 31–985-062 |
| Mixed cellulose membrane | EMD Millipore | Cat# GSWP02500 |
| Streptavidin biosensors | Molecular Devices | Cat# 18–5019 |
| Sf-900 III SFM | Thermo Fisher Scientific | Cat# 12,658,027 |
| OptiPlate-384, White Opaque 384-well Microplate | PerkinElmer | Cat# 6,007,290 |
| Greiner 384well, black, flat bottom polypropylene plates | Millipore Sigma | Cat# M1937–32EA |
| 96-well Flat Clear Bottom Black Polystyrene Microplates | Corning | Cat# 3340 |
| poly-D-lysine-coated white, clear-bottom 96-well assay plates | Greiner Bio-One | Cat# 655,944 |
| White Adhesive Bottom Seal | Perkin Elmer | Cat# 6,005,199 |
| Dounce tissue grinder set | Millipore Sigma | Cat# D8938–1SET |
| Spark 20 M plate reader | TECAN | N/A |
| Synergy H4 Hybrid Microplate Reader | BioTek | N/A |
| Octet RED384 | ForteBio | N/A |
| LS 6500 Multi-Purpose Scintillation Counter | Beckman Coulter | N/A |
| Axopatch 200B amplifier | Molecular Devices | N/A |
| Digidata 1550B digitizer | Molecular Devices | N/A |
| Sutter P-97 puller | Sutter Instrument Company | N/A |
| Syro Wave automated peptide synthesizer | Biotage | N/A |