Research ArticleGPCR SIGNALING

The GPCR accessory protein MRAP2 regulates both biased signaling and constitutive activity of the ghrelin receptor GHSR1a

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Science Signaling  07 Jan 2020:
Vol. 13, Issue 613, eaax4569
DOI: 10.1126/scisignal.aax4569
  • Fig. 1 MRAP2 potentiates the ghrelin-stimulated and inhibits the constitutive activity of GHSR1a.

    (A) Ghrelin-stimulated IP accumulation in CHO cells expressing GHSR1a with empty vector or increasing amount of MRAP2. Results are shown in fold increase over baseline and represent the means + SEM of three independent experiments performed in triplicate. (B) Ghrelin-stimulated IP accumulation in CHO cells expressing GHSR1a with empty vector or MRAP2 at a 1:20 ratio. Results are shown as the percentage of the maximal response in cells expressing GHSR1a and empty vector and represent the means + SEM of three independent experiments performed in triplicate. (C) Measurement of IP accumulation over 1 hour in the presence of lithium and absence of agonist in cells transfected with increasing amount of GHSR1a and either empty vector or MRAP2. Empty vector and MRAP2 concentration were kept constant. Data represent the means + SEM of three independent experiments performed in triplicate. (D) Ghrelin-stimulated IP accumulation in CHO cells expressing GHSR1a(A204E) with empty vector or MRAP2. Measurement of IP3 production over 1 hour in the presence of lithium and absence of agonist in cells transfected with GHSR1a and empty vector, MRAP2, or the indicated MRAP2 mutant. Error bars are means ± SEM. Data represent the results of at least three independent experiments. Statistical analysis was done using multiple t test (one unpaired t test per condition). ***P < 0.001, *P < 0.05.

  • Fig. 2 MRAP2 inhibits β-arrestin recruitment to GHSR1a.

    (A) CHO cells transfected with plasmids encoding 3HA-GHSR1a and mCherry–β-arrestin2 with or without plasmid encoding MRAP2 were analyzed by immunofluorescence microscopy. Micrographs of live cells were taken 10 min after stimulation with vehicle or 100 nM ghrelin. mCherry signal is shown in red, and nuclei stained with Hoechst are shown in blue. Scale bars, 10 μm. Images are representative of three separate experiments. (B) Schematic of the NanoBiT-based β-arrestin recruitment assay. Cells express GHSR1a-LgBiT and SmlBiT–β-arrestin. Agonist-stimulated recruitment of β-arrestin allows the recombination of a functional NanoLuc enzyme, resulting in measurable luminescent signal. (C) Kinetics of ghrelin-stimulated β-arrestin recruitment in cells expressing GHSR1a alone (blue) or with MRAP2 (red). The blue arrow indicates the time at which ghrelin was injected. Data are the means ± SEM of three independent experiments performed in triplicate. T test comparing the area under the curve was used for statistical analysis. (D) Surface density of GHSR1a measured by fixed-cell ELISA. Data are the means ± SEM of three independent experiments performed in triplicate, and significance was measured by one-way analysis of variance (ANOVA). EV, empty vector. (E) Concentration-response curve of ghrelin-stimulated β-arrestin recruitment to GHSR1a in the presence or absence of MRAP2. Statistical analysis of the differences at each ghrelin concentration was measured by t test. Data are means ± SEM of three independent experiments performed in triplicate. ***P < 0.001.

  • Fig. 3 Regulation of G protein– and β-arrestin–dependent signaling pathways by MRAP2.

    (A) Schematic of CRISPR-Cas9 targeted deletion for Arrb1 and Arrb2 genes. (B) PCR validation of β-arrestin1 KO, β-arrestin2 KO, and β-arrestin1/β-arrestin2 double KO cell lines. (C and D) Concentration response curves of ghrelin-stimulated IP3 production in WT (C) and β-arrestin1/2 KO (D) cells transfected with GHSR1a with or without MRAP2. Data represent the means ± SEM of three independent experiments performed in triplicate. (E and F) Concentration response curves of ghrelin-stimulated RhoA activation in WT (E) and β-arrestin1/2 KO (F) CHO cells transfected with SRF-RE-Luc reporter, GHSR1a, and either empty vector or MRAP2. Statistical analysis of the differences at each ghrelin concentration was measured by t test. Data represent the means ± SEM of three independent experiments performed in triplicate. ***P < 0.001.

  • Fig. 4 Identification of the regions of MRAP2 required for the potentiation of G protein–dependent GHSR1a signaling.

    (A) Schematic representation of the different MRAP2 mutants generated. TM, transmembrane domain. (B) Western blot detection of MRAP2 mutants from lysates of transfected CHO cells (n = 3 independent experiments). (C to H) Ghrelin-stimulated IP accumulation in CHO cells transfected with GHSR1a and empty vector, MRAP2, or the indicated MRAP2 mutant. Statistical analysis of the differences at each ghrelin concentration was measured by t test. Data represent the means ± SEM of three independent experiments performed in triplicate. ***P < 0.001.

  • Fig. 5 Identification of MRAP2 regions required for the inhibition of β-arrestin recruitment to GHSR1a.

    (A to F) Kinetics of ghrelin-stimulated β-arrestin recruitment to GHSR1a in CHO cells transfected with GHSR1a-LgBiT, SmlBiT–β-arrestin2, and empty vector, MRAP2, or the indicated MRAP2 mutant. Statistical analysis of the differences at each ghrelin concentration was measured by t test. Data represent the means ± SEM of three independent experiments performed in triplicate. (G) Surface density of GHSR1a measured by fixed-cell ELISA in each of the condition tested for β-arrestin recruitment. Statistical analysis was done using one-way ANOVA. Data represent the means ± SEM of three independent experiments performed in triplicate. (H) Area under the curve of β-arrestin recruitment normalized to the surface expression of GHSR1a for each condition tested. Statistical analysis was done using one-way ANOVA. Data are shown as means ± SEM of the percentage of normalized β-arrestin recruitment in the absence of MRAP2. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 6 Identification of MRAP2 regions required for the inhibition of ghrelin-stimulated RhoA activation.

    (A to F) Luminescence reporting ghrelin-stimulated RhoA activity in CHO cells expressing the SRF-RE reporter, GHSR1a, and either empty vector, MRAP2, or the indicated MRAP2 mutant. Statistical analysis of the differences at each ghrelin concentration was measured by t test. Data are shown in fold increase over baseline and are means ± SEM of three experiments performed in triplicate. ***P < 0.001.

  • Fig. 7 MRAP2 does not increase ghrelin affinity or Gαq protein activation.

    (A) 125I-Ghrelin competition binding assay in CHO cells transfected with GHSR1a and empty vector or MRAP2. Nonspecific binding from mock-transfected cells was subtracted, and results were normalized to the maximal binding for each condition. Data shown are the means ± SEM of three independent experiments performed in triplicate. (B) GTPγS binding assay in membranes prepared from CHO cells transfected with GHSR1a and empty vector or MRAP2. Binding values obtained with membranes from mock-transfected cells were subtracted, and results were normalized to GHSR1a expression measured by ELISA. Statistical analysis was done using one-way ANOVA. Results shown represent the data attained from three independent experiments. Error bars are SEM. ***P < 0.001, **P < 0.01.

Supplementary Materials

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    • Fig. S1. Surface expression of WT and mutated MRAP2.

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