Research ArticleGPCR SIGNALING

Preassembled GPCR signaling complexes mediate distinct cellular responses to ultralow ligand concentrations

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Science Signaling  09 Oct 2018:
Vol. 11, Issue 551, eaan1188
DOI: 10.1126/scisignal.aan1188
  • Fig. 1 GPCRs respond to subnanomolar concentrations of ligand.

    (A to C) Quantification of cAMP in native HEK293 cells stimulated with increasing concentrations of adenosine, the βAR agonist Iso, or PGE1 (A); the muscarinic acetylcholine receptor agonist CCh, the δ-opioid receptor agonist SNC80, or dopamine (B); and relaxin or GLP-1 (C) in the absence of the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) (n = 6 to 9 independent experiments; see also table S1). (D and E) Quantification of cAMP in native CHO-K1 cells stimulated with increasing concentrations of adenosine or PGE1 (D) and 5-HT or thrombin (E) in the presence of IBMX (n = 6 independent experiments). (F) Quantification of cAMP in native HEK293 cells stimulated with increasing concentrations of adrenaline, noradrenaline, or acetylcholine in the absence of IBMX (n = 6 to 8 independent experiments; see also table S1). (G) Quantification of cAMP in primary human cardiac fibroblasts (CFs) stimulated with increasing concentrations of Iso or CCh in the absence of IBMX (n = 5 to 6 independent experiments). (H) Quantification of cAMP in native HEK293 cells or HEK293 cells transiently expressing scrambled or β2AR small interfering RNA (siRNA), stimulated with increasing concentrations of Iso in the absence of IBMX (n = 6 independent experiments). (I) Expression of β2AR mRNA in native HEK293 cells or HEK293 cells transiently expressing scrambled (scram.) or β2AR siRNA as determined by qRT-PCR (n = 3 independent experiments). (J) Quantification of cAMP in native HEK293 cells or HEK293 cells transiently expressing scrambled or M3R siRNA, stimulated with increasing concentrations of CCh in the absence of IBMX (n = 6 independent experiments). (K) Expression of M3R mRNA in native HEK293 cells or HEK293 cells transiently expressing scrambled or M3R siRNA as determined by qRT-PCR (n = 3 independent experiments). (L) Quantification of cAMP in HEK293 cells transiently expressing the β2AR or M3R and stimulated with increasing concentrations of Iso or CCh, respectively, in the absence of IBMX (n = 3 to 4 independent experiments). All data are expressed as the means ± SEM of n independent experiments. *P < 0.05 and **P < 0.01 versus HEK293 controls, one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test.

  • Fig. 2 Femtomolar concentrations of ligand cause sustained increases in plasma membrane–localized cAMP and require an intact orthosteric binding site and only one binding event per cell.

    (A) Measurement of cAMP at the plasma membrane in single native HEK293 cells using the FRET biosensor pmEpac2, which reversibly binds cAMP. Cells were stimulated with vehicle, 1 fM Iso, or 100 nM Iso (n = 47 to 79 cells). (B) Representative ratiometric pseudocolor images of cells from (A) at the indicated time points after stimulation. Scale bars, 10 μm. (C) Measurement of cAMP at the plasma membrane in single native HEK293 cells preincubated with the β2AR antagonist ICI-118,551 before stimulation with vehicle or 1 fM Iso (n = 51 to 97 cells). (D) Measurement of cAMP at the plasma membrane in single native HEK293 cells stimulated with vehicle, 1 fM CCh, or 1 μM CCh (n = 29 to 53 cells). (E) Representative ratiometric pseudocolor images of cells from (D) at the indicated time points after stimulation. Scale bars, 10 μm. (F) Measurement of cAMP at the plasma membrane in single native HEK293 cells preincubated with the M3R antagonist NMS before stimulation with vehicle or 1 fM CCh (n = 56 to 95 cells). (G) Measurement of cAMP at the plasma membrane in single HEK293 cells transiently expressing wild-type (WT) FLAG-β2AR or the orthosteric binding site D3.32A mutant FLAG-β2AR and stimulated with vehicle, 1 fM Iso, or 1 pM Iso (n = 43 to 151 cells). (H) Measurement of cAMP at the plasma membrane in single HEK293 cells transiently expressing WT or D3.32A mutant 3HA-M3R and stimulated with vehicle, 1 fM CCh, or 1 pM CCh (n = 119 to 186 cells). (I) Measurement of cAMP at the plasma membrane in single HEK293 cells transiently expressing M3R-DREADD and stimulated with vehicle, 1 fM CCh, or 1 fM CNO (n = 57 to 89 cells). All cells were stimulated at 0 min, and a maximal cAMP response (Max) was induced after 5 min by stimulating the cells with forskolin, IBMX, and PGE1. Individual cells were analyzed from experiments performed on three independent occasions. Data are expressed as the means ± SEM of n cells, normalized to the maximal cAMP response induced after 5 min (F/FMax). (J) Fraction of HEK293 cells within the field of view that increased cAMP at the plasma membrane after a 5-min exposure to 1 fM or 100 nM Iso. Data were analyzed from experiments in Fig. 3 (A and B) with an area under the curve (AUC) greater than 0.697 considered statistically significantly increased compared to vehicle control. Data are expressed as the means ± SEM of six independent experiments. (K) The 95% credible interval for responses to 1 fM Iso over 5 min, using 1000 randomly subsampled parameter sets from the Markov chain Monte Carlo (MCMC) sampling procedure. The red line shows the time course with parameters consistent with the maximum a posteriori probability (MAP) estimate. The solid gray line shows the median, and the dashed gray lines show the 95% credible interval for the subsampled parameter sets. The 1 fM Iso data from (J) is shown as crosses; for two of these, only a small region (~2%) of sampled parameter space allows the model to reach these points. (L) Normalized frequency of binding for 1 fM Iso from 100 independent model simulations with the MAP estimate parameter set. The average number of binding events is 1.13 per cell.

  • Fig. 3 A preassembled β2AR signaling complex controls the response to femtomolar concentrations of ligand.

    (A) Measurement of cAMP at the plasma membrane in response to 5 min of stimulation with vehicle or 1 fM Iso in single native HEK293 cells that were pretreated with the Gαs antagonist NF449, the Gβγ inhibitor mSIRK, the negative control peptide mSIRK L9A, or the AC inhibitor ddA or transient expression of scrambled, AKAP250, β-arrestin 1, or β-arrestin 2 siRNA (n = 36 to 254 cells). (B) Measurement of cAMP at the plasma membrane in response to 5 min of stimulation with vehicle or 1 fM Iso in single native HEK293 cells was pretreated with the Gαi/o antagonist NF023, the PDE inhibitor IBMX, or the PKA inhibitor KT5720 or transient expression of PDE4D3 dominant negative (dn), PDE4D5 dn, pSilencer control, or AKAP79 short hairpin RNA (shRNA) (n = 22 to 254 cells). (C) Measurement of cAMP at the plasma membrane after 5 min of stimulation with vehicle or 1 fM Iso in HEK293 cells transiently expressing the β2AR. Cells were pretreated with the Gαi/o antagonist NF023, the PDE inhibitor IBMX, or the PKA inhibitor KT5720 or transient coexpression of PDE4D3 dn, PDE4D5 dn, pSilencer control, or AKAP79 shRNA (n = 22 to 153 cells). All cells (A to C) were stimulated at 0 min, and a maximal cAMP response was induced after 5 min by the addition of forskolin, IBMX, and PGE1. Individual cells were analyzed from experiments performed on three independent occasions. Data are expressed as the means ± SEM of n cells and represented as the 5-min AUC. **P < 0.01 and ***P < 0.001 versus vehicle control, two-way ANOVA with Sidak’s multiple comparison test; ^P < 0.05, ^^P < 0.01, and ^^^P < 0.001 versus untreated control, two-way ANOVA with Dunnett’s multiple comparison test. (D) Cartoon showing the regions of the β2AR C-terminal tail (CT) that were tagged with glutathione S-transferase (GST). (E) Quantification of proteins identified as required for activation of cAMP in response to 1 fM Iso in GST pulldowns from lysates of unstimulated native HEK293 cells using the indicated immobilized CT-GST fusions. GST pulldowns were assayed for endogenous Gαs (short and long forms), transgenically expressed AC2-HA, endogenous β-arrestin 1, and endogenous β-arrestin 2 (n = 5 to 6). (F) Quantification of proteins identified as required for regulation of constitutive activity of the preassembled β2AR complex in GST pulldowns from lysates of unstimulated native HEK293 cells using the indicated CT-GST fusions. GST pulldowns were assayed for endogenous Gαi in cells transgenically expressing AKAP79-HA, endogenous PKA, transgenically expressed PDE4D5 dn, and transgenically expressed AKAP79-HA (n = 3 to 4). For GST pulldown assays (E to F), band densities were normalized for equivalent amounts of GST and expressed relative to GST alone. Data are means ± SEM of n independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 versus GST alone, two-way ANOVA with Dunnett’s multiple comparison test. (G) Representative immunoblots (IB) showing Gαs, β-arrestin 1, β-arrestin 2, PKA, PDE4D, HA, and Gαi in GST pulldown assays of lysates from cells using GST alone or the indicated CT-GST fusions. (H) Representative immunoblots showing β2AR and HA after HA immunoprecipitation (IP) of lysates from HEK293 cells transiently expressing HA-AKAP250. (I) Representative images of cells coexpressing β2AR-CFP and a YFP-tagged component of the β2AR-associated complex or the positive control pmEpac2, after acceptor photobleaching of a region of the plasma membrane (dotted box). Gray solid boxes indicate areas of the plasma membrane that were photobleached previously. Scale bars, 10 μm. (J) FRET efficiency at the plasma membrane between β2AR-CFP and YFP-tagged components of the protein complex, calculated from acceptor photobleaching FRET experiments from two regions of interest (ROIs) per cell with four cells analyzed per biological replicate (n = 24 ROIs). Data are expressed as the means ± SEM of n ROIs. *P < 0.05 and ***P < 0.001 versus β2AR-CFP/Gαq-YFP FRET efficiency, Kruskal-Wallis with Dunn’s multiple comparison test; ^^P < 0.01 and ^^^P < 0.001 versus β2AR-CFP/Gαq-YFP FRET after conversion to binary values (1 = FRET, 0 = no FRET) and then chi-square test. (K) Cartoon of the preassembled β2AR signaling complex required for responses to femtomolar concentrations of Iso. Stimulation of cells with 1 fM Iso activates a Gαs- and Gβγ-mediated stimulation of AC2 that depends on AKAP250 and β-arrestins 1 and 2. This increase in cAMP causes the sequential activation of PKA and PDE4D5, which cooperates with Gαi/o to oppose the increase in cAMP. This tonic opposition depends on AKAP79. Hierarchy of proteins within the cartoon is based on whether proteins mediate activation or inhibition and reported protein-protein interactions (3, 5, 53, 59, 84, 105).

  • Fig. 4 A preassembled M3R signaling complex controls the response to femtomolar concentrations of ligand.

    (A) Measurement of cAMP at the plasma membrane in response to 5 min of stimulation with vehicle or 1 fM CCh in single native HEK293 cells that were pretreated with the Gαs antagonist NF449, the Gαq/11 inhibitor UBO-QIC, the Gβγ inhibitor mSIRK, the negative control peptide mSIRK L9A, the PKC inhibitor GF109203X, or the AC inhibitor ddA or transiently transfected with scrambled, AKAP250, β-arrestin 1 siRNA, or β-arrestin 2 siRNA (n = 39 to 316 cells). (B) Measurement of cAMP at the plasma membrane in response to 5 min of stimulation with vehicle or 1 fM CCh in single native HEK293 cells that were pretreated with the Gαi/o antagonist NF023, the PDE inhibitor IBMX, or the PKA inhibitor KT5720 or transiently transfected with PDE4D3 dn, PDE4D5 dn, pSilencer control, or AKAP79 shRNA (n = 31 to 316 cells). (C) Measurement of cAMP at the plasma membrane after 5 min of stimulation with vehicle or 1 fM CCh in HEK293 cells transiently expressing the M3R. Cells were pretreated with the Gαi/o antagonist NF023, the PDE inhibitor IBMX, or the PKA inhibitor KT5720 or transiently cotransfected with PDE4D3 dn, PDE4D5 dn, pSilencer control, or AKAP79 shRNA (n = 65 to 193 cells). All cells (A to C) were stimulated at 0 min, and a maximal cAMP response was induced after 5 min with forskolin, IBMX, and PGE1. Individual cells were analyzed from experiments performed on three independent occasions. Data are expressed as the means ± SEM of n cells, and represented as the 5 min AUC. **P < 0.01 and ***P < 0.001 versus vehicle control, two-way ANOVA with Sidak’s multiple comparison test; ^^P < 0.01 and ^^^P < 0.001 versus untreated control, two-way ANOVA with Dunnett’s multiple comparison test. (D) Cartoon showing the regions of the M3R third intracellular loop (ICL3) that were tagged with GST. (E) Quantification of proteins required for activation of cAMP in response to 1 fM CCh in GST pulldowns from unstimulated native HEK293 cells using the indicated GST-ICL3 fusions. GST pulldowns were assayed for endogenous Gαq/11, endogenous PKC (from cells transgenically expressing with AKAP79-HA), transgenically expressed AC2-HA, endogenous β-arrestin 1, endogenous β-arrestin 2, and transgenically expressed AKAP79-HA (n = 3 and 4). (F) Quantification of GST pulldowns from unstimulated native HEK293 cell lysates of proteins required for regulation of constitutive activity of the preassembled M3R complex: endogenous PKA and transgenically expressed PDE4D3 dn (n = 3 to 4). For GST pulldown assays, band densities were normalized for equivalent amounts of GST and expressed relative to GST alone. Data are means ± SEM of n independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 versus GST alone, two-way ANOVA with Dunnett’s multiple comparison test. (G) Representative immunoblots showing Gαq/11, PKC, HA, β-arrestin 1, β-arrestin 2, PKA, and PDE4D in GST pulldown assays of lysates using GST alone or the indicated ICL3-GST fusions. (H) Representative images of cells coexpressing M3R-CFP and a YFP-tagged component of the M3R protein complex or the positive control pmEpac2, after acceptor photobleaching of a region of the plasma membrane (dotted box). Scale bars, 10 μm. (I) FRET efficiency at the plasma membrane between M3R-CFP and YFP-tagged components of the protein complex, calculated from acceptor photobleaching FRET experiments from two ROIs per cell with four cells analyzed per biological replicate (n = 24 ROIs). Data are expressed as the means ± SEM of n ROIs. *P < 0.05 and ***P < 0.001 versus M3R-CFP/Gαs-YFP FRET efficiency, Kruskal-Wallis with Dunn’s multiple comparison test; ^P < 0.05 and ^^^P < 0.001 versus M3R-CFP/Gαs-YFP FRET after conversion to binary values (1 = FRET, 0 = no FRET) and then chi-square test. (J) Cartoon of the preassembled M3R signaling complex required for responses to femtomolar concentrations of CCh. Stimulation of cells with 1 fM CCh activates a Gαq/11-Gβγ-PKC–mediated stimulation of AC2 that depends on AKAP79 and β-arrestins 1 and 2. This increase in cAMP causes the sequential activation of PKA and PDE4D3, which opposes the increase in cAMP. Hierarchy of proteins within the cartoon is based on reported protein-protein interactions (5, 55, 64).

  • Fig. 5 Stimulation of the β2AR and M3R by femtomolar concentrations of ligand activates sustained and compartmentalized kinase signaling.

    (A to F) Single native cells were stimulated with vehicle or the indicated concentration of Iso for 20 min. (A) ERK activity detected in the nucleus of HEK293 cells using the FRET biosensor, EKAR, which is reversibly phosphorylated by ERK and targeted to the nucleus (nucEKAR) (n = 118 to 133 cells). Data are normalized to the maximal ERK response (F/FMax). (B) Representative ratiometric pseudocolor images of cells from (A) at the indicated time points after stimulation. Scale bars, 10 μm. (C) ERK activity detected in the cytosol using the cytoEKAR FRET biosensor or nucleus (nucEKAR) of HEK293 cells. Some cells were stimulated with 1 fM CCh instead of Iso for 20 min (n = 13 to 130 cells). Data are represented as the 20-min AUC. (D) ERK activity detected in the nucleus of human CFs (n = 38 to 61 cells). Data are normalized to the baseline ERK response (F/F0). (E) cAMP detected at the plasma membrane in HEK293 cells (n = 31 to 44 cells). Data are normalized to the maximal cAMP response induced after 20 min (F/FMax). (F) cAMP detected at the plasma membrane of human CFs (n = 22 to 53 cells). Data are normalized to the baseline cAMP response (F/F0). (G to L) Single native cells were stimulated with vehicle or the indicated concentration of CCh for 20 min. (G) PKC activity detected in the cytosol of HEK293 cells using the FRET biosensor, CKAR, which is reversibly phosphorylated by PKC (n = 185 to 226 cells). Data are normalized to the maximal PKC response induced after 20 min (F/FMax). (H) Representative ratiometric pseudocolor images of cells from (G) at the indicated time points after stimulation. Scale bars, 10 μm. (I) PKC activity detected at the plasma membrane (pmCKAR) or in the cytosol (cytoCKAR) of HEK293 cells. Some cells were stimulated with 1 fM Iso instead of CCh (n = 10 to 175 cells). Data are represented as the 20-min AUC. (J) PKC activity detected in the cytosol of human CFs (n = 69 to 124 cells). Data are normalized to the baseline PKC response (F/F0). (K) cAMP detected at the plasma membrane of HEK293 cells (n = 32 to 44 cells). Data are normalized to the maximal cAMP response induced after 20 min (F/FMax). (L) cAMP detected at the plasma membrane of human CFs (n = 31 to 50 cells). Data are normalized to the baseline cAMP response (F/F0). All cells were stimulated at 0 min, and a maximal ERK, PKC, or cAMP response was induced after 20 min with PDBu (ERK), PDBu plus phosphatase inhibitors (PKC), or forskolin plus IBMX and PGE1 (cAMP). Individual cells were analyzed from experiments performed on three independent occasions. Data are expressed as the means ± SEM of n cells. ***P < 0.001 versus vehicle control, two-way ANOVA with Sidak’s multiple comparison test.

  • Fig. 6 Activation of the β2AR and M3R by femtomolar concentrations of ligand causes distinct whole-cell responses.

    (A) Representative hierarchical clustering of proteins with increased (blue) or decreased (red) abundance in native HEK293 cell populations after stimulation with vehicle, 1 fM or 100 nM Iso, or 1 fM or 10 μM CCh for 4 hours. Data are expressed as Z-scores of the ligand-stimulated log2 change in protein abundance compared to vehicle (see also table S3) (n = 3 independent experiments). (B) Proteins with a statistically significant increase or decrease in abundance in native HEK293 cells after stimulation with Iso or CCh were classified by Gene Ontology (GO) terms and grouped into the indicated categories. A Biological Process GO term was included if it occurred in at least two of the three independent experiments. (C) Log2 change in protein abundance in the indicated native HEK293 treatment groups versus vehicle control for SF3B5, TXNL4A, RPS21, GUF1, and TXNDC9, all of which are involved in RNA processing and protein synthesis (n = 3). (D and E) GFP fluorescence in single native HEK293 cells expressing the pEF1α-GFP reporter after stimulation with vehicle, 1 fM or 100 nM Iso (D; n = 196 to 204 cells), or 1 fM or 10 μM CCh (E; 177 to 194 cells) for 4 hours. Individual cells were analyzed from three independent experiments. Data are expressed relative to baseline fluorescence (F/F0). (F) GFP fluorescence in single human CFs expressing the pEF1α-GFP reporter after stimulation with vehicle, 1 fM Iso, or 100 nM Iso for 4 hours (n = 64 to 107 cells). Individual cells were analyzed from four independent experiments. Data are expressed as relative fluorescence units (RFU) per cell. (G) GFP fluorescence in single human CFs expressing the pEF1α-GFP reporter after stimulation with vehicle, 1 fM CCh, or 10 μM CCh (n = 109 to 121 cells) and activation of Cdc42, as measured by the Raichu-Cdc42 FRET biosensor, which detects GDP/GTP binding, in single human CFs after stimulation with vehicle, 1 fM Iso, or 100 nM Iso (n = 133 to 178 cells) expressed as the 4-hour AUC. (H) Log2 change in protein abundance in the indicated native HEK293 treatment groups versus vehicle control for GGA1, PDE6D, ILK, VPS52, and GPSM1, all of which are involved in protein trafficking and cytoskeletal networks (n = 3). (I and J) Activation of Cdc42 in single native HEK293 cells after stimulation with vehicle, 1 fM or 10 μM CCh (I; n = 305 to 323 cells), or 1 fM or 100 nM Iso (J; n = 304 to 401 cells) for 4 hours. Individual cells were analyzed from three independent experiments. Data are expressed relative to baseline FRET (F/F0). (K) Activation of Cdc42 in single human CFs after stimulation with vehicle, 1 fM CCh, or 10 μM CCh for 4 hours (n = 150 to 159 cells). Individual cells were analyzed from three independent experiments. Data are expressed relative to baseline FRET (F/F0). All data are expressed as the means ± SEM of n cells or independent experiments. **P < 0.01 versus vehicle control, two-way ANOVA with Dunnett’s multiple comparison test (C and H). ***P < 0.001 versus vehicle control, two-way ANOVA (D, F, I, and K).

  • Fig. 7 GPCR signaling complexes respond to femtomolar concentrations of ligand.

    GPCRs exist in preassembled protein complexes at the plasma membrane. (1) Simulation of stochastic ligand-receptor binding kinetics reveals that the addition of a 1 fM solution of ligand under our assay conditions would result in an average of one to two binding events per cell within 5 min. (2) One to two binding events stimulate strong signal amplification, which depends on a preassembled protein complex at the plasma membrane and results in (3) a relatively slow and gradual increase in the signal over time. (4) Addition of a high concentration solution of ligand (100 nM Iso or 1 μM CCh) results in a much greater number of binding events and activates receptors that are present in preassembled complexes and in any uncomplexed receptors. (5) The resulting activation stimulates a signal that is qualitatively different from that elicited by ultralow ligand concentrations, such as (6) no signal (CCh-stimulated cAMP, EF1α gene transcription, or Cdc42 activity) or (7) a more rapid increase in the signal that then declines (Iso-stimulated cAMP, nuclear ERK, or cytosolic PKC).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/551/eaan1188/DC1

    Fig. S1. Endogenous expression of transcripts encoding GPCRs in HEK293 cells.

    Fig. S2. Biphasic changes in cAMP are due to activation of endogenous β2AR and M3R.

    Fig. S3. FRET biosensors can detect responses of endogenous or exogenous receptors to femtomolar concentrations of ligand.

    Fig. S4. Plasma membrane localization and activity of mutant β2AR and M3R.

    Fig. S5. Modeling responses to femtomolar concentrations of Iso.

    Fig. S6. Identification of proteins involved in mediating responses to 1 fM Iso.

    Fig. S7. The β2AR forms a preassembled signaling complex.

    Fig. S8. Identification of proteins involved in mediating responses to 1 fM CCh.

    Fig. S9. The M3R forms a preassembled signaling complex.

    Fig. S10. Femtomolar ligand concentrations activate compartmentalized signaling and unique cell responses.

    Table S1. Femtomolar concentrations of ligand increase cAMP.

    Table S2. The β2AR and M3R constitutively form complexes at the plasma membrane.

    Table S3. Proteins from hierarchical clustering analysis.

  • The PDF file includes:

    • Fig. S1. Endogenous expression of transcripts encoding GPCRs in HEK293 cells.
    • Fig. S2. Biphasic changes in cAMP are due to activation of endogenous β2AR and M3R.
    • Fig. S3. FRET biosensors can detect responses of endogenous or exogenous receptors to femtomolar concentrations of ligand.
    • Fig. S4. Plasma membrane localization and activity of mutant β2AR and M3R.
    • Fig. S5. Modeling responses to femtomolar concentrations of Iso.
    • Fig. S6. Identification of proteins involved in mediating responses to 1 fM Iso.
    • Fig. S7. The β2AR forms a preassembled signaling complex.
    • Fig. S8. Identification of proteins involved in mediating responses to 1 fM CCh.
    • Fig. S9. The M3R forms a preassembled signaling complex.
    • Fig. S10. Femtomolar ligand concentrations activate compartmentalized signaling and unique cell responses.
    • Legends for tables S1 to S3

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Femtomolar concentrations of ligand increase cAMP.
    • Table S2 (Microsoft Excel format). The β2AR and M3R constitutively form complexes at the plasma membrane.
    • Table S3 (Microsoft Excel format). Proteins from hierarchical clustering analysis.

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