Research ArticleGPCR SIGNALING

Distinct profiles of functional discrimination among G proteins determine the actions of G protein–coupled receptors

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Science Signaling  01 Dec 2015:
Vol. 8, Issue 405, pp. ra123
DOI: 10.1126/scisignal.aab4068
  • Fig. 1 Fingerprinting GPCR activity by measuring signaling efficacy and kinetics across a set of G proteins.

    (A) Schematic representation of the BRET assay. Activation of a GPCR by agonist leads to the dissociation of inactive heterotrimeric G proteins into active GTP-bound Gα and Venus-Gβγ subunits. The free Venus-Gβγ then interacts with the Gβγ effector mimetic masGRK3ct-Nluc to produce the BRET signal. (B) Representative response profile showing the BRET signal generated by the D2 dopamine receptor in the presence of Gαo. Dopamine (100 μM) was applied to the cells, and six independent reactions were conducted in parallel. (C) Quantification of response variability between the different indicated sensors. (D) Repertoire of mammalian Gα subunits. G proteins marked in red were successfully reconstituted in the NanoBRET system. Scale bar below represents relative evolutionary distance. (E to I) Fingerprinting responses of the M3R to the physiological ligand ACh. (E) Quantification of the maximal response amplitudes generated by the M3R. The maximum amplitudes from the 14 different G proteins were normalized to the largest value to obtain comparative agonist efficacy and were plotted at corresponding vertices in the wheel diagram. The thickness of the lines connecting each data point represents the SEM of four experiments performed in parallel. (F) Dose-response curve of two representative signaling pathways in response to Ach. Data are means ± SEM of four experiments. (G) Quantification of the G protein activation rates catalyzed by the M3R. Activation rate constants from 14 different G proteins were normalized to the response that produced the maximum value and are plotted for each of the G proteins tested. Data are means ± SEM of four experiments. (H) Comparison of the time courses of activation of GoA and Gq. Each trace represents the mean of the responses measured in eight wells. (I) Maximal response amplitudes recorded at different times [1 and 5 s marked in (H)] after agonist application. Data are means ± SEM of four experiments.

  • Fig. 2 Characteristic profiles of G protein activation distinguish various GPCRs from each other.

    Several GPCRs that belong to different subfamilies were examined for the specificity of their G protein coupling by measuring two parameters: maximum amplitude of the BRET signal (red) and activation rates (blue). Cells expressing the M3R, β2AR, BDKB2R, or D2R were activated by saturating concentrations (100 μM) of their respective endogenous agonists: ACh, adrenaline, bradykinin, and dopamine. The data reflecting maximum BRET amplitude and activation rate are plotted as relative activity values after normalization against the G protein species that exhibited maximal activity. Data are means ± SEM of four to six experiments.

  • Fig. 3 Major classes of intracellular G protein regulators have distinct effects on GPCR fingerprints.

    (A) The G protein coupling profiles of the M3 receptor were examined in cells in the absence of regulatory molecules (left), in the presence of RGS8 (middle), or in the presence of AGS1 (right). Data are means ± SEM of four experiments. (B and C) Effect of RGS8 on the deactivation rates of Go and G15. Cells were pretreated with 100 μM ACh for 35 s and then were treated with 1 mM atropine (a muscarinic antagonist). Traces correspond to the deactivation phase of the responses of GoA (B) and G15 (C) in the absence and presence of RGS8 and are the average of 12 experiments, normalized to the response at the time of atropine application. (D) The deactivation rate constants in the absence (black) or presence (red) of RGS8 were measured for all responding G proteins. Data are means ± SEM of four experiments. (E and F) Effect of AGS1 on the activation of GoB and Gq. Cells were cotransfected with plasmids encoding M3R (E and F) and either GoB (E) or Gq (F) with (red) or without (black) plasmid encoding AGS1. BRET signals before (basal) and after the application of Ach were recorded. Each trace is an average of six replicates. (G) Quantification of changes in the basal BRET ratio for the indicated G proteins measured in the absence (black) or presence (red) of AGS1. Data are means ± SEM of six experiments. The unpaired t test was used to test for statistically significant differences between untransfected cells and RGS8-expressing (D) or AGS1-expressing (G) cells. *P < 0.001.

  • Fig. 4 Synthetic GPCR ligands can bias the G protein coupling profiles of GPCRs.

    (A) Four different agonist application conditions (yellow boxes) were examined for their effects on the G protein fingerprints of the M1R using two parameters: maximum amplitude (red) and activation rates (blue). Saturating concentrations (100 μM) of ACh, OXO-M, TBPB, or ACh and TBPB were applied to the M1R-expressing cells. Data are means ± SEM of six experiments. (B and C) Individual comparison of the activation of GoA (B) and Gq (C) by ACh (black) or TBPB (red). Each trace represents the mean of 12 replicates. (D) Direct comparison of the effects of the indicated agonists on amplitudes of the responses of GoA and Gq to M1R. (E) Direct comparison of the effects of the indicated agonists on the activation kinetics of GoA and Gq by M1R. Data are means ± SEM of six replicates. *P < 0.001 by paired t test. #P < 0.01 by paired t test. N.D., not detected.

  • Fig. 5 Ligand-dependent coupling of muscarinic receptors to GIRK channels in native hippocampal neurons.

    (A) Schematic representation of the activation of GIRK channels by GPCRs. The binding of agonist to a Gi/o-coupled GPCR leads to an interaction between Gβγ and the GIRK channel, which evokes an inwardly rectifying K+ current. (B) Representative traces of GIRK currents in hippocampal neurons evoked by a saturating concentration (100 μM) of the indicated agonists. (C) Maximal current amplitudes of GIRK responses elicited by agonist were measured 10 s after agonist application. The application of TBPB either in the absence or presence of OXO-M did not evoke any inward current. (D) Current densities in the presence of a high concentration of K+ were measured to assess ligand-independent ion flow through inwardly rectifying potassium channels. The amount of current was recorded before the application of each indicated agonist. All electrophysiological data were recorded from a total of seven neurons. Data are means ± SEM.

  • Fig. 6 GPCR fingerprinting reveals the selective activation of G proteins by opioid receptors in response to a classical antagonist.

    (A to F) Endogenous agonists (endomorphin-1 or dynorphin A) and a classical antagonist (naloxone) were examined for their effects on the G protein coupling specificities of MOR (A to C) and KOR (D to F) using two parameters: maximum amplitude (red) and activation rates (blue). Saturating concentrations (100 μM) of the indicated ligands were applied. Data are means ± SEM of 6 to 12 experiments. (B, C, E, and F) Direct comparison of the activation of GoA (black) and Gi1 (red) by MOR (B and C) and KOR (E and F) in response to endomorphin-1 (B), dynorphin A (E), or naloxone (C and F). Each trace represents an average of six replicates.

  • Fig. 7 The biased G protein coupling specificities of opioid receptor subtypes in response to naloxone results in differential modulation of cAMP production.

    (A) Schematic representation of the assay paradigm. Transfected cells expressing opioid receptors were preincubated with naloxone before the β2AR agonist Iso was applied to stimulate cAMP production. The kinetics of the amplitude of the cAMP signal were determined in real time with a BRET-based cAMP sensor that exhibits a decreased BRET signal upon cAMP binding. (B to E) Effect of naloxone on Iso-stimulated cAMP production in HEK 293T/17 cells expressing no opioid receptor (B), MOR (C), KOR (D), or DOR (E). The cells were cotransfected with plasmids encoding the indicated opioid receptors together with Nluc-Epac-VV. Before the activation of endogenous β2ARs with Iso, transfected cells were incubated with (closed circle) or without (open circles) 100 μM naloxone for 5 min. The cells were then treated with 1 μM Iso at time zero. Each trace represents the mean of 12 replicates. (F) Quantification of changes in maximal BRET amplitudes induced by naloxone for each of the opioid receptors. §P < 0.05 and *P < 0.0001 by paired t test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/405/ra123/DC1

    Fig. S1. Characterization of the performance of the Nluc-based BRET assay.

    Fig. S2. Effects of Ric-8A and Ric-8B on the expression of Gα subunits and their responsiveness to agonist.

    Fig. S3. Optimization of the stoichiometry of Gα and Venus-Gβγ.

    Fig. S4. Traces showing real-time activity measurements of 14 different G proteins.

    Fig. S5. The plasma membrane localization of Venus-Gβγ is dependent on coexpression with Gα subunits to ensure 1:1 complex stoichiometry.

    Fig. S6. Exogenous GPCR and Gα stimulate BRET responses in the assay.

    Fig. S7. The abundances of heterotrimers of Gα and Venus-Gβγ in cells transiently transfected with plasmids encoding 14 different Gα subunits are similar.

    Fig. S8. GPCR responses are within the dynamic range of the assay.

    Fig. S9. Fingerprinting of the deactivation phase.

    Fig. S10. Direct comparison of the agonist-induced coupling of M1R to different G proteins.

    Fig. S11. Characterization of DOR fingerprints.

  • Supplementary Materials for:

    Distinct profiles of functional discrimination among G proteins determine the actions of G protein–coupled receptors

    Ikuo Masuho, Olga Ostrovskaya, Grant M. Kramer, Christopher D. Jones, Keqiang Xie, Kirill A. Martemyanov

    *Corresponding author. E-mail: kirill{at}scripps.edu

    This PDF file includes:

    • Fig. S1. Characterization of the performance of the Nluc-based BRET assay.
    • Fig. S2. Effects of Ric-8A and Ric-8B on the expression of Gα subunits and their responsiveness to agonist.
    • Fig. S3. Optimization of the stoichiometry of Gα and Venus-Gβγ.
    • Fig. S4. Traces showing real-time activity measurements of 14 different G proteins.
    • Fig. S5. The plasma membrane localization of Venus-Gβγ is dependent on coexpression with Gα subunits to ensure 1:1 complex stoichiometry.
    • Fig. S6. Exogenous GPCR and Gα stimulate BRET responses in the assay.
    • Fig. S7. The abundances of heterotrimers of Gα and Venus-Gβγ in cells transiently transfected with plasmids encoding 14 different Gα subunits are similar.
    • Fig. S8. GPCR responses are within the dynamic range of the assay.
    • Fig. S9. Fingerprinting of the deactivation phase.
    • Fig. S10. Direct comparison of the agonist-induced coupling of M1R to different G proteins.
    • Fig. S11. Characterization of DOR fingerprints.

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    Citation: I. Masuho, O. Ostrovskaya, G. M. Kramer, C. D. Jones, K. Xie, K. A. Martemyanov, Distinct profiles of functional discrimination among G proteins determine the actions of G protein–coupled receptors. Sci. Signal. 8, ra123 (2015).

    © 2015 American Association for the Advancement of Science

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