Research ArticlePharmacology

G protein subtype–specific signaling bias in a series of CCR5 chemokine analogs

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Science Signaling  16 Oct 2018:
Vol. 11, Issue 552, eaao6152
DOI: 10.1126/scisignal.aao6152
  • Fig. 1 CCR5-mediated Gi/o protein activation by endogenous chemokines and RANTES analogs.

    (A) The RANTES analogs used in this study have modifications to the N-terminal tail region as shown, where p represents pyroglutamate, α represents N-nonanoyl, β represents l-thioproline, and γ represents l-2-cyclohexylglycine. (B) Human embryonic kidney (HEK) 293T cells cotransfected with CCR5 and the EPAC2 reporter plasmid were stimulated with forskolin and incubated with the chemokines RANTES (red, circle), MIP-1α (black, square), 5P12 (blue, diamond), 5P14 (green, hexagon), 6P4 (yellow, triangle), and PSC (purple, star). (C) Transfected cells were pretreated with PTX (100 ng/ml; squares) or control buffer (circles) for 16 hours, stimulated with forskolin, and incubated with 100 nM chemokines. Data are expressed as a percentage of forskolin-stimulated response and are presented as the mean ± SEM for n ≥ 3 three independent experiments performed in at least technical triplicate. Shown are the statistical significance of the differences between PTX-treated and control condition for each chemokine: ****P < 0.0001 (multiple t tests). Each chemokine tested significantly inhibited cAMP generation (P < 0.001). PSC and 6P4 showed enhanced inhibition of forskolin-induced cAMP generation [P < 0.05, one-way analysis of variance (ANOVA), Dunnett’s multiple comparisons test].

  • Fig. 2 The effects of G protein–subtype cotransfection on CCR5-mediated Ca2+ flux and IP1 accumulation in response to endogenous chemokines and RANTES analogs.

    Ca2+ flux in HEK293T cells transfected with (A) CCR5 alone or with G protein α subunits (B) Gi2, (C) Gq, or (D) Gqi5 was measured in response to 100 nM chemokine. Fluorescence data (maximal minus basal) are presented as mean relative fluorescence units (RFU) ± SEM for n = 4 independent experiments performed in technical duplicate. IP1 accumulation in HEK293T cells transfected with (E) CCR5 alone or with G protein α subunits (F) Gi2, (G) Gq, or (H) Gqi5 was measured in response to 100 nM chemokine. Data are expressed as picomoles of IP1 formed per well and are presented as the mean ± SEM for n = 4 independent experiments performed in technical duplicate. Shown are the statistical significance of the differences between chemokine treatment and buffer control and between chemokine treatment and RANTES treatment: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA, Tukey’s multiple comparisons test).

  • Fig. 3 The effects of PTX and YM-254890 treatment on CCR5-mediated Ca2+ flux in response to endogenous chemokines and RANTES analogs.

    Ca2+ flux in HEK293T cells transfected with (A) CCR5 alone or with G protein α subunits (B) Gi2, (C) Gq, or (D) Gqi5 was measured in response to 100 nM chemokine. Cells were pretreated with buffer (white bars), PTX (100 ng/ml; gray bars), or 1 μM YM-254890 (black bars). Fluorescence data (maximal minus basal) are presented as mean RFU ± SEM for n = 4 independent experiments performed in technical duplicate. Shown are the statistical significance of the differences between buffer control, PTX, and YM-254890: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA, Tukey’s multiple comparisons test).

  • Fig. 4 The effects of PTX and YM-254890 treatment on CCR5-mediated IP1 accumulation in response to endogenous chemokines and RANTES analogs.

    IP1 accumulation in HEK293T cells transfected with (A) CCR5 alone or with G protein α subunits (B) Gi2, (C) Gq, or (D) Gqi5 was measured in response to 100 nM chemokine. Cells were pretreated with buffer (white bars), PTX (100 ng/ml; gray bars), or 1 μM YM-254890 (black bars). Data are expressed as picomoles of IP1 formed per well and are presented as the mean ± SEM for n = 4 independent experiments performed in technical duplicate. Shown are the statistical significances of the differences between buffer control, PTX, and YM-254890: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA, Tukey’s multiple comparisons test).

  • Fig. 5 Dose-response curves for CCR5-mediated Ca2+ flux and IP1 accumulation with or without cotransfection of different G protein α subunits.

    Ca2+ flux in HEK293T cells transfected with (A) CCR5 alone or with G protein α subunits (B) Gi2, (C) Gq, or (D) Gqi5 was measured in response to RANTES (red, circle), MIP-1α (black, square), 5P12 (blue, diamond), 5P14 (green, hexagon), 6P4 (yellow, triangle), and PSC (purple, star). Fluorescence data (maximal minus basal) are presented as mean RFU ± SEM for n = 4 independent experiments performed in technical triplicate. IP1 accumulation in HEK293T cells transfected with (E) CCR5 alone or with G protein α subunits (F) Gi2, (G) Gq, or (H) Gqi5 was measured in response to RANTES (red, circle), MIP-1α (black, square), 5P12 (blue, diamond), 5P14 (green, hexagon), 6P4 (yellow, triangle), and PSC (purple, star). Data are expressed as picomoles of IP1 formed per well and are presented as the mean ± SEM for n = 3 independent experiments performed in technical triplicate.

  • Fig. 6 Conceptual model depicting CCR5-mediated signaling through Gi/o and Gq by endogenous chemokines and RANTES analogs.

    The CCR5 receptor exists in at least two populations, with a group of CCR5 molecules precoupled to Gi/o (gray background) and another group in an uncoupled or naked state (blue background). (A) RANTES and MIP-1α bind only to Gi/o-precoupled CCR5, which then leads to activation of Gi/o. After activation of Gi/o, CCR5 can then recruit and activate Gq. 6P4 and PSC bind to both Gi/o-precoupled CCR5 and naked CCR5. Thus, activation of Gq protein occurs independently of Gi/o proteins. (B) 5P12 binds to both Gi/o-precoupled CCR5 and naked CCR5, but binding leads to activation of Gi/o only.

  • Table 1 Summary of fitted curve parameters for inhibition of cAMP accumulation.

    EC50, pEC50, and Emax values with SEM are given for Fig. 1B.

    RANTESMIP-1α5P125P146P4PSC
    CCR5
      EC50 (nM)0.712.10.40.10.4
      pEC50 ± SEM−9.2 ± 0.19−9.0 ± 0.26−8.7 ± 0.38−9.4 ± 0.22−9.9 ± 0.31−9.4 ± 0.56
      Emax ± SEM26 ± 2.320 ± 2.518 ± 3.231 ± 3.120 ± 3.014 ± 3.4
  • Table 2 Summary of fitted curve parameters for Ca2+ flux.

    EC50, pEC50, and Emax values with SEM are given for Fig. 5, left column. ND, not detectable.

    RANTESMIP-5P125P146P4PSC
    CCR5
      EC50 (nM)32240ND213625
      pEC50 ± SEM−7.6 ± 0.2−6.5 ± 0.2ND−7.7 ± 0.7−7.4 ± 0.1−7.6 ± 0.1
      Emax ± SEM12,000 ± 100015,000 ± 2000ND3000 ± 100045,000 ± 200031,000 ± 2000
    CCR5 + Gi2
      EC50 (nM)NDNDNDND11042
      pEC50 ± SEMNDNDNDND−6.9 ± 0.3−7.4 ± 0.3
      Emax ± SEMNDNDNDND15,000 ± 300010,000 ± 2000
    CCR5 + Gq
      EC50 (nM)32460NDND2126
      pEC50 ± SEM−7.5 ± 0.1−6.3 ± 0.3NDND−7.7 ± 0.1−7.6 ± 0.1
      Emax ± SEM20,000 ± 200027,000 ± 8000NDND61,000 ± 400064,000 ± 2000
    CCR5 + Gqi5
      EC50 (nM)0.95.1233.60.71.8
      pEC50 ± SEM−9.0 ± 0.1−8.3 ± 0.1−7.6 ± 0.1−8.4 ± 0.1−9.1 ± 0.1−8.7 ± 0.1
      Emax ± SEM50,000 ± 400050,000 ± 300019,000 ± 100045,000 ± 200077,000 ± 400067,000 ± 3000
  • Table 3 Summary of fitted curve parameters for IP1 accumulation.

    EC50, pEC50, and Emax values are given for Fig. 5, right column. ND, not detectable.

    RANTESMIP-5P125P146P4PSC
    CCR5
      EC50 (nM)NDNDNDNDND190
      pEC50 ± SEMNDNDNDNDND−6.7 ± 0.4
      Emax ± SEMNDNDNDNDND1.7 ± 0.3
    CCR5 + Gi2
      EC50 (nM)NDNDNDNDNDND
      pEC50 ± SEMNDNDNDNDNDND
      Emax ± SEMNDNDNDNDNDND
    CCR5 + Gq
      EC50 (nM)65NDND4.31872
      pEC50 ± SEM−7.2 ± 0.4NDND−8.3 ± 0.3−7.7 ± 0.1−7.1 ± 0.1
      Emax ± SEM1.0 ± 0.2NDND1.6 ± 0.37.4 ± 0.18.4 ± 0.1
    CCR5 + Gqi5
      EC50 (nM)62.90.50.63.525
      pEC50 ± SEM−8.2 ± 0.1−8.5 ± 0.1−9.3 ± 0.4−9.2 ± 0.2−8.5 ± 0.1−7.6 ± 0.1
      Emax ± SEM3.1 ± 0.33.0 ± 0.22.3 ± 0.45.3 ± 0.45.8 ± 0.55.4 ± 0.3

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/552/eaao6152/DC1

    Fig. S1. Response of cells transfected with EPAC reporter and vector to chemokines and RANTES analogs.

    Fig. S2. Dose-response curves for CCR5-mediated Gi/o protein activation by chemokines and RANTES analogs using cells expressing higher amounts of CCR5.

    Fig. S3. The effect of chemokines and RANTES analogs on cAMP accumulation in the presence of PTX.

    Fig. S4. Time courses of CCR5-mediated inhibition of cAMP accumulation induced by chemokines and RANTES analogs.

    Fig. S5. Response of vector-transfected cells to chemokines and RANTES analogs through stimulation of Ca2+ flux.

    Fig. S6. The effect of G protein subunit cotransfection on the amount of CCR5 molecules on the cell surface.

    Fig. S7. ATP-induced Ca2+ flux in HEK293T cells expressing CCR5 and G protein subunits.

    Fig. S8. The effect of G protein subunit cotransfection on CCR5-mediated Ca2+ flux and IP1 accumulation in response to chemokines and RANTES analogs.

    Fig. S9. IP1 accumulation in vector-transfected cells in response to chemokines and RANTES analogs.

    Fig. S10. Dose-response curves for CCR5-mediated Ca2+ flux in response to chemokines and RANTES analogs using cells expressing higher amounts of CCR5.

  • This PDF file includes:

    • Fig. S1. Response of cells transfected with EPAC reporter and vector to chemokines and RANTES analogs.
    • Fig. S2. Dose-response curves for CCR5-mediated Gi/o protein activation by chemokines and RANTES analogs using cells expressing higher amounts of CCR5.
    • Fig. S3. The effect of chemokines and RANTES analogs on cAMP accumulation in the presence of PTX.
    • Fig. S4. Time courses of CCR5-mediated inhibition of cAMP accumulation induced by chemokines and RANTES analogs.
    • Fig. S5. Response of vector-transfected cells to chemokines and RANTES analogs through stimulation of Ca2+ flux.
    • Fig. S6. The effect of G protein subunit cotransfection on the amount of CCR5 molecules on the cell surface.
    • Fig. S7. ATP-induced Ca2+ flux in HEK293T cells expressing CCR5 and G protein subunits.
    • Fig. S8. The effect of G protein subunit cotransfection on CCR5-mediated Ca2+ flux and IP1 accumulation in response to chemokines and RANTES analogs.
    • Fig. S9. IP1 accumulation in vector-transfected cells in response to chemokines and RANTES analogs.
    • Fig. S10. Dose-response curves for CCR5-mediated Ca2+ flux in response to chemokines and RANTES analogs using cells expressing higher amounts of CCR5.

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