Research ArticleGPCR SIGNALING

Type 2 diabetes–associated variants of the MT2 melatonin receptor affect distinct modes of signaling

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Science Signaling  28 Aug 2018:
Vol. 11, Issue 545, eaan6622
DOI: 10.1126/scisignal.aan6622
  • Fig. 1 Functional profiling of MT2 variants for spontaneous receptor activity.

    (A) Spontaneous Gαi1 activity of WT MT2 and the indicated MT2 variants as measured by BRET. (B) Spontaneous Gαz activity of WT MT2 and the indicated MT2 variants as measured by BRET. (C) Spontaneous recruitment of β-arrestin2 (β-arr2) to WT MT2 and the indicated MT2 variants as measured by the PathHunter β-arrestin2 assay. Data are shown as a percentage of the activity of WT MT2 (indicated by the dotted line) and are means ± SEM of at least three experiments. One-sample t test was performed to assess statistically significant differences between the WT receptor and the MT2 variants. *P < 0.05, **P < 0.01, and ***P < 0.0001. See also tables S1 to S5 for complete data sets for the spontaneous activity of MT2 variants.

  • Fig. 2 MLT concentration-response curves for the MT2-mediated signaling of variants representing each signaling profile.

    (A to G) Analysis of the signaling profiles of MT2 variants. The MT2 variant tested is listed on the left. Each of the five categories of signaling profile is listed above the row of graphs for each variant receptor. The effect of each variant on signaling is listed for each panel. Solid lines with filled circles correspond to the variant MT2, and dotted lines with open circles correspond to the WT MT2, which was monitored in parallel with the variant receptors in each experiment. Data were plotted using nonlinear regression with a variable Hill slope. Data are means ± SEM of at least three experiments. See also fig. S4 and tables S1 to S5 for complete data sets for the agonist-mediated signaling activity of MT2 variants.

  • Fig. 3 Relationships between spontaneous and MLT-induced MT2 variant activation, between G protein– and β-arrestin2–dependent events, and between different G protein signaling events.

    (A) Correlation plots between the spontaneous and MLT-induced effects (Emax) of each variant MT2 for Gαi1 activation (left), Gαz activation (middle), and β-arrestin2 recruitment (right). (B) Correlation plots between the Emax values of MLT-induced Gαi1 activation, Gαz activation, and cAMP inhibition with β-arrestin2 recruitment for each variant MT2. (C) Correlation plots between the Emax values of MLT-induced Gαi1activation and Gαz activation (left), Gαi1 activation and cAMP inhibition (middle), and Gαz activation and cAMP inhibition of each variant MT2. Spontaneous activities and Emax values represent means ± SEM of at least three experiments and were normalized to those of the WT MT2. Data were fitted by linear regression analysis, and R2 values were obtained for the overall correlation. All R values are statistically significant with P values <0.0001. See also fig. S6 for correlations between spontaneous and agonist-promoted activity for G proteins and β-arrestin signaling parameters.

  • Fig. 4 Graphical representation of the signaling signatures of MT2 variants.

    Radial graphs representing spontaneous and MLT-induced activation of Gαi1 and Gαz, inhibition of cAMP production, recruitment of β-arrestin2, and activation of ERK by WT MT2 and the 40 indicated MT2 variants. The activity of WT MT2 was set as zero. Values of variants with enhanced properties ranged from 0 to +1, and those with impaired properties ranged from 0 to −1. The scale of all radial graphs ranged from −1 to +1. S, spontaneous; Em, agonist-mediated efficacy; Δ, Δlog(τ/KA). See also tables S1 to S5 for complete data sets for spontaneous and agonist-mediated signaling activity of the MT2 variants.

  • Fig. 5 Evolutionary action analysis of MT2 variants and correlation with signaling impairment.

    (A) An evolutionary action score was calculated for each indicated MT2 variant. EA ranges from 0 to 100, with a score of 0 predicted as benign and a score of 100 predicted as highly impactful or detrimental to protein function. Scores are colored on the basis of EA [EA = 0 (pink) to EA = 100 (blue)]. (B) Functional defects of every MT2 variant were defined by phenotype score and correlated with the EA score. Data were fitted by linear regression analysis, and R2 values were obtained for the overall correlation. Correlation value was statistically significant, P < 0.0001. The A42P MT2 variant was excluded from the R2 calculation because of the known detrimental effect of proline residues on general protein structure. See also tables S1 to S5 for complete data sets for spontaneous and agonist-mediated signaling activity of the MT2 variants.

  • Table 1 Association of specific functional defects of rare MT2 variants with T2D risk.

    Only rare MT2 variants (with a frequency <1%) were included. By applying Bonferroni correction, a statistically significant P value was <0.0056. A P value between 0.05 and 0.0056 was considered a trend of association (a nominal association). Nine independent association tests were performed on the basis of a T2D case-control study including 2186 individuals with T2D and 4804 controls. CI, confidence interval.

    Consequences on MT2 functionRare MT2 VariantsFrequency (cases)Frequency
    (controls)
    Odds ratio
    (95% CI)
    P value
    Defects in Gαi1
    activation
    SpontaneousA25T, A42P, L60R,
    P95L, M120I/V,
    S123R, V124I,
    R138C/H/L, Y141F,
    T201M, I223T, F250V,
    Y308S, R316H
    1.1%0.7%2.32 (1.17–4.60)0.016
    MLT-inducedA42P, L60R, A74T,
    P95L, S123R, V124I,
    R138C/H/L, Y141F,
    R154H, T201M,
    R222H, I223T, F250V,
    Y308S, R316H,
    R330W, A342V
    1.4%0.7%3.25 (1.73–6.10)2.4 × 10−4
    Defects in Gαz
    activation
    SpontaneousA42P, L60R, P95L,
    M120V, S123R,
    R138C/H/L, T201M,
    R222H, I223T, F250V,
    Y308S, R316H
    0.9%0.5%2.95 (1.35–6.46)0.0067
    MLT-inducedA42P, L60R, A74T,
    P95L, G109A,
    M120I/V, S123R,
    V124I, R138C/H/L,
    Y141F, T201M,
    R222H, I223T,
    D246N, F250V,
    Y308S, R316H,
    R330W, A342V
    1.4%0.8%2.93 (1.57–5.45)7.3 × 10−4
    Defects in cAMP
    inhibition
    MLT-inducedA42P, L60R, P95L,
    G109A, M120V,
    S123R, R138C/H/L,
    R222H, I223T, F250V,
    Y308S, R316H,
    R330W
    0.9%0.6%2.83 (1.31–6.12)0.0083
    Defects in β-arrestin2
    recruitment
    SpontaneousA42P, L60R, P95L,
    G109A, S123R, V124I,
    R138C/L, Y141F,
    R154H, R222H, I223T,
    F250V, Y308S, A342V
    1.3%0.7%3.06 (1.59–5.90)8.3 × 10−4
    MLT-inducedA42P, L60R, A74T,
    P95L, G109A, M120V,
    S123R, V124I,
    R138C/H/L, L166I,
    T201M, R222H,
    I223T, R231H, E237K,
    S238G, D246N,
    F250V, Y308S,
    R316H, A342V
    2.7%2.3%2.13 (1.23–3.70)0.0071
    Defects in ERK
    activation
    MLT-inducedA42P, L60R, A74T,
    P95L,M120I, S123R,
    V124I, R138C/H/L,
    M146V, R154H,
    T201M, R222H,
    I223T, F250V, Y308S,
    R316H
    1.2%0.8%2.49 (1.29–4.80)0.0066
    Neutral variantsA8S, A13V, G21S,
    W22L, A25T, P36S,
    A52T, A234T, I353T,
    A359E
    0.2%0.2%2.50 (0.59–10.59)0.21

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/545/eaan6622/DC1

    Fig. S1. Screening for G proteins and detection of pathways activated by WT MT2.

    Fig. S2. Predicted topology and distribution of the 40 MT2 variants.

    Fig. S3. Detection of MT2 variants at the cell surface.

    Fig. S4. MLT dose-response curves for Gαi1 and Gαz activation, β-arrestin2 recruitment, cAMP inhibition, and ERK activation by MT2 variants.

    Fig. S5. G protein–dependent but β-arrestin–independent MLT-mediated ERK phosphorylation by MT2.

    Fig. S6. Correlations between the effects of the mutations on spontaneous and agonist-promoted activity for G proteins and β-arrestin signaling parameters performed pairwise.

    Table S1. Summary of the functional profiling of Gαi1 activation by WT MT2 and MT2 variants.

    Table S2. Summary of the functional profiling of Gαz activation by WT MT2 and MT2 variants.

    Table S3. Summary of the functional profiling of β-arrestin2 recruitment to WT MT2 and MT2 variants.

    Table S4. Summary of the functional profiling of cAMP inhibition by WT MT2 and MT2 variants.

    Table S5. Summary of the functional profiling of ERK activation by WT MT2 and MT2 variants.

  • This PDF file includes:

    • Fig. S1. Screening for G proteins and detection of pathways activated by WT MT2.
    • Fig. S2. Predicted topology and distribution of the 40 MT2 variants.
    • Fig. S3. Detection of MT2 variants at the cell surface.
    • Fig. S4. MLT dose-response curves for Gαi1 and Gαz activation, β-arrestin2 recruitment, cAMP inhibition, and ERK activation by MT2 variants.
    • Fig. S5. G protein–dependent but β-arrestin–independent MLT-mediated ERK phosphorylation by MT2.
    • Fig. S6. Correlations between the effects of the mutations on spontaneous and agonist-promoted activity for G proteins and β-arrestin signaling parameters performed pairwise.
    • Table S1. Summary of the functional profiling of Gαi1 activation by WT MT2 and MT2 variants.
    • Table S2. Summary of the functional profiling of Gαz activation by WT MT2 and MT2 variants.
    • Table S3. Summary of the functional profiling of β-arrestin2 recruitment to WT MT2 and MT2 variants.
    • Table S4. Summary of the functional profiling of cAMP inhibition by WT MT2 and MT2 variants.
    • Table S5. Summary of the functional profiling of ERK activation by WT MT2 and MT2 variants.

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