Research ArticleDevelopmental Biology

G protein–coupled receptors control the sensitivity of cells to the morphogen Sonic Hedgehog

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Science Signaling  06 Feb 2018:
Vol. 11, Issue 516, eaao5749
DOI: 10.1126/scisignal.aao5749
  • Fig. 1 GPR161 suppresses the potency of Hedgehog ligands in NIH/3T3 cells.

    (A) Immunoblot showing the indicated proteins in extracts from Sonic Hedgehog (SHH)–treated wild-type (WT) NIH/3T3 cells and four clonal Gpr161−/− NIH/3T3 cell lines (P1 to P4) generated using four different guide RNAs. GLI3FL, full-length GLI3; GLI3R, GLI3 transcriptional repressor fragment. (B) Quantification of Gli1 mRNA relative to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in WT and four independently generated Gpr161−/− NIH/3T3 cell lines after exposure to the indicated range of SHH concentrations. EC50, concentration that produces a half-maximal response. AU, arbitrary units. (C) Immunoblot showing the indicated proteins in extracts from WT and two different Gpr161−/− NIH/3T3 cell lines (P1 and P2) that were either untreated or treated with increasing concentrations of SHH. (D) SHH-induced activation of a luciferase-based Hedgehog (Hh) reporter gene in NIH/3T3 cells transiently transfected with an empty vector or a vector carrying a gene encoding GPR161. (E and F) Quantification of Gli1 mRNA relative to Gapdh by qRT-PCR in WT or Gpr161−/− NIH/3T3 cells after exposure to the indicated range of SHH concentrations in the presence or absence of small molecules that inhibit Smoothened (SMO) [cyclopamine (E)] or GPCR kinase 2 (GRK2) [Takeda compound 101 (cmpd101) (F)]. In (B) and (D) to (F), each data point represents a mean of three technical replicates. (A), (B), (E), and (F) show representative data from three independent experiments. (C) and (D) show representative data from two independent experiments.

  • Fig. 2 GRK2 functions at a step between SMO and Gαs in the Hh signaling pathway.

    (A) The current model for transduction of Hh signals in vertebrates, with positive regulators in green and negative regulators in red. Glioma-associated oncogene (GLI) proteins (yellow) can function as either transcriptional repressors or activators, depending on how they are posttranslationally modified and processed. SMO is thought to reduce protein kinase A (PKA) activity by reducing the amount of GPR161 in primary cilia. Shown to the right of each component are the results from genetic epistasis experiments to establish the order in which the component functions relative to GRK2. SUFU, Suppressor of Fused; PTCH, patched proteins. (B to E) Quantification of endogenous Gli1 mRNA relative to Gapdh by qRT-PCR in cells of various genotypes that were exposed to the indicated combinations of SHH and the GRK2 inhibitor cmpd101. In (B), each data point represents mean ± SD (n = 3). In (C) to (E), each data point represents mean ± SD (n = 4). Statistical significance was determined by unpaired Welch’s t test and depicted as follows: **P < 0.01, ***P < 0.001, ****P < 0.0001, and P > 0.05. n.s., not significant.

  • Fig. 3 Mapping the residues that are critical for GRK2 function in Hh signaling.

    (A) Domain structure of GRK2 [adapted from (59)]. RH, regulator of G protein (heterotrimeric guanine nucleotide–binding protein) signaling homology; AGC C-tail, C-terminal extension of the kinase domain reminiscent of PKA, PKG, and PKC; PH, pleckstrin homology. Amino acid residues mutated in our analysis are indicated, along with their previously established effects on GRK2 function. (B) Activation of a luciferase-based Hh reporter gene in Grk2−/− NIH/3T3 cells in response to SHH after transient transfection with an empty vector or a vector carrying genes encoding the indicated mutant forms of GRK2–green fluorescent protein (GFP). Each data point represents mean ± SD (n = 3). Statistical significance was determined by unpaired Welch’s t test and depicted as follows: **P < 0.01.

  • Fig. 4 GRK2 and GRK3 activity is required for spinal neural cell fates that are dependent on all levels of Hh signaling.

    (A) A schematic of the progenitor domains within the embryonic spinal cord [adapted from (36)]. NC, notochord; FP, floor plate; pMN, motor neuron progenitors; p0, p1, p2, and p3, ventral interneuron progenitors. The progenitor domains are a product of a high-to-low SHH gradient (pink) along the ventral-to-dorsal axis. The bars on the right represent the transcription factors present in each progenitor domain. (B) Neural progenitor cells (NPCs) carrying the fluorescent Hh reporter GLI binding site (GBS)–Venus were left untreated or treated with SHH, cmpd101, or SHH + cmpd101, followed by immunofluorescence staining to count the percentage of cells positive for transcription factors that define ventral progenitor subtypes summarized in (A). Each data point represents the data from one image of an NPC colony (see fig. S4A) consisting of 100 to 200 cells, and each condition is represented by 15 different colonies. The experiment was repeated twice. Medians with interquartile ranges are shown with statistical significance determined by the Mann-Whitney nonparametric analysis of variance (ANOVA) test and depicted as follows: ****P < 0.0001. (C and D) Immunoblots (representative of two independent experiments) showing the indicated proteins in WT, Grk2−/− (C), or Grk2−/− Grk3−/− double-null NPCs (D) treated with the indicated combinations of SHH and cmpd101. Two Grk2−/− Grk3−/− clonal NPC lines (A1 and A4) are shown in (D), and an independent Grk2−/− NPC cell line is shown in fig. S4C.

  • Fig. 5 GPR161 suppresses low-level Hh responses and attenuates high-level Hh responses in NPCs.

    (A) Hh signaling was assessed (n = 3 independent experiments) using immunoblots of extracts from WT and Gpr161−/− NPCs left untreated or treated with SHH. (B) Activation of the GBS-Venus reporter in WT or Gpr161−/− NPCs treated with increasing concentrations of SHH. Each data point represents median reporter fluorescence calculated from 10,000 cells. (C) A schematic (left) of the progenitor domains within the embryonic spinal cord along with opposing gradients of GLI repressor (GLIR) and GLI activator (GLIA) proteins proposed to establish the spatial pattern of neural subtypes. Pax6, Nkx6.1, Olig2, Nkx2.2, and Foxa2 mRNAs were quantified by qRT-PCR (normalized to Gapdh) in WT and Gpr161−/− NPCs treated with increasing concentrations of SHH. Immediately to the left of each graph, the domains in the neural tube where each transcription factor is present are depicted as a color code based on the diagram to the left. Each data point represents a mean of three technical replicates. The experiments in (B) and (C) were repeated twice.

  • Fig. 6 GRK2 and SMO are required for high-level Hh responses in Gpr161−/− NPCs.

    (A) A schematic of the progenitor domains, with reciprocal GLIR and GLIA domains, as previously shown in Fig. 5C. (B and C) The percentage of cells containing progenitor subtype markers (NKX6.1, OLIG2, NKX2.2, and GBS-Venus) with or without SHH exposure in Gpr161−/− NPCs assessed at 48 hours after inhibition of GRK2/3 with cmpd101 (B) or inhibition of SMO with cyclopamine (C). Each data point represents the data from one image of an NPC colony (see fig. S6A) consisting of 100 to 200 cells, and each condition is represented by 15 different colonies. The experiment was repeated twice. Medians with interquartile ranges are shown with statistical significance determined by the Mann-Whitney nonparametric ANOVA test and depicted as follows: ****P < 0.0001 and P > 0.05. (D) Summary of the effect of GRK2 and SMO inhibitors on the differentiation of WT and Gpr161−/− NPCs.

  • Fig. 7 S negatively regulates all levels of Hh signaling in NPCs.

    (A) Immunoblot (representative of three independent experiments) showing the effect of GRK2/3 inhibition (by cmpd101) on SHH-induced responses in WT and Gnas−/− NPCs. FOXA2 abundance is driven by the highest level of Hh signaling during neural tube development and, hence, can be considered a marker of maximal Hh signaling in this system. (B) A model for the different consequences of GPR161 loss in NIH/3T3 cells and NPCs. Decreasing PKA activity drives increasing levels of signaling, first by preventing GLIR formation and subsequently by allowing formation of GLIA. We speculate that the decrease in PKA activity (arrows) produced by the loss of GPR161 is greater in NPCs compared to NIH/3T3 cells (top). In NPCs only, this decrease in PKA activity drops below the threshold required for the biogenesis of GLI3R and consequently allows for the adoption of cell fates (bottom; NKX6.1- and OLIG2-positive cells) repressed by GLI3R in the basal state. In both NPCs and NIH/3T3 cells, the lowered PKA activity sensitizes cells to SHH-induced responses, shifting the SHH dose-response curve to the left.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/516/eaao5749/DC1

    Fig. S1. Cmpd101 inhibits Hh signaling in NIH/3T3 cells.

    Fig. S2. GRK2 affects the ciliary localization of GPR161 in NIH/3T3 cells.

    Fig. S3. GRK2 functions at a step between SMO and GαS in Hh signaling.

    Fig. S4. SHH signaling in NPCs is mediated through GRK2/3.

    Fig. S5. GPR161 suppresses NPC differentiation responses that are driven by low-level Hh signals.

    Fig. S6. High-level Hh responses in Gpr161−/− NPCs depend on GRK2 and SMO.

    Fig. S7. GαS regulates both low-level and high-level Hh responses in NPCs.

    Table S1. List of pooled or clonal knockout cell lines generated by CRISPR/Cas9.

  • Supplementary Materials for:

    G protein–coupled receptors control the sensitivity of cells to the morphogen Sonic Hedgehog

    Ganesh V. Pusapati, Jennifer H. Kong, Bhaven B. Patel, Mina Gouti, Andreas Sagner, Ria Sircar, Giovanni Luchetti, Philip W. Ingham, James Briscoe, Rajat Rohatgi*

    *Corresponding author. Email: rrohatgi{at}stanford.edu

    This PDF file includes:

    • Fig. S1. Cmpd101 inhibits Hh signaling in NIH/3T3 cells.
    • Fig. S2. GRK2 affects the ciliary localization of GPR161 in NIH/3T3 cells.
    • Fig. S3. GRK2 functions at a step between SMO and GαS in Hh signaling.
    • Fig. S4. SHH signaling in NPCs is mediated through GRK2/3.
    • Fig. S5. GPR161 suppresses NPC differentiation responses that are driven by low-level Hh signals.
    • Fig. S6. High-level Hh responses in Gpr161−/− NPCs depend on GRK2 and SMO.
    • Fig. S7. GαS regulates both low-level and high-level Hh responses in NPCs.
    • Table S1. List of pooled or clonal knockout cell lines generated by CRISPR/Cas9.

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    Citation: G. V. Pusapati, J. H. Kong, B. B. Patel, M. Gouti, A. Sagner, R. Sircar, G. Luchetti, P. W. Ingham, J. Briscoe, R. Rohatgi, G protein–coupled receptors control the sensitivity of cells to the morphogen Sonic Hedgehog. Sci. Signal. 11, eaao5749 (2018).

    © 2018 American Association for the Advancement of Science

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