Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Feb 6;11(516):eaao5749.
doi: 10.1126/scisignal.aao5749.

G Protein-Coupled Receptors Control the Sensitivity of Cells to the Morphogen Sonic Hedgehog

Affiliations
Free PMC article

G Protein-Coupled Receptors Control the Sensitivity of Cells to the Morphogen Sonic Hedgehog

Ganesh V Pusapati et al. Sci Signal. .
Free PMC article

Abstract

The morphogen Sonic Hedgehog (SHH) patterns tissues during development by directing cell fates in a concentration-dependent manner. The SHH signal is transmitted across the membrane of target cells by the heptahelical transmembrane protein Smoothened (SMO), which activates the GLI family of transcription factors through a mechanism that is undefined in vertebrates. Using CRISPR-edited null alleles and small-molecule inhibitors, we systematically analyzed the epistatic interactions between SMO and three proteins implicated in SMO signaling: the heterotrimeric G protein subunit GαS, the G protein-coupled receptor kinase 2 (GRK2), and the GαS-coupled receptor GPR161. Our experiments uncovered a signaling mechanism that modifies the sensitivity of target cells to SHH and consequently changes the shape of the SHH dose-response curve. In both fibroblasts and spinal neural progenitors, the loss of GPR161, previously implicated as an inhibitor of basal SHH signaling, increased the sensitivity of target cells across the entire spectrum of SHH concentrations. Even in cells lacking GPR161, GRK2 was required for SHH signaling, and Gαs, which promotes the activation of protein Kinase A (PKA), antagonized SHH signaling. We propose that the sensitivity of target cells to Hedgehog morphogens, and the consequent effects on gene expression and differentiation outcomes, can be controlled by signals from G protein-coupled receptors that converge on Gαs and PKA.

Conflict of interest statement

Competing Interests: PWI owns a small amount of stock shares in Curis, Inc., a biotechnology company with interests in pharmacologically targeting Hedgehog signaling.

Figures

Fig. 1
Fig. 1. GPR161 suppresses the potency of Hh ligands in NIH/3T3 cells.
(A) Immunoblot showing the indicated proteins in extracts from SHH-treated wild-type NIH/3T3 cells and four clonal Gpr161-/- NIH/3T3 cell lines (P1-P4) generated using four different guide RNAs. (B) Quantification of Gli1 mRNA relative to Gapdh by qRT-PCR in wild-type (WT) and four independently generated Gpr161-/- NIH/3T3 cell lines after exposure to the indicated range of SHH concentrations. (C) Immunoblot showing the indicated proteins in extracts from wild-type 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 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 wild-type or Gpr161-/- NIH/3T3 cells after exposure to the indicated range of SHH concentrations in the presence or absence of small molecules that inhibit SMO (cyclopamine, E) or GRK2 (cmpd101, F). In B, D, E and 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
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. GLI proteins (yellow) can function as either transcriptional repressors or activators, depending on how they are post-translationally modified and processed. SMO is thought to reduce 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. (B-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±S.D (n=3). In C to E, each data point represents mean±S.D (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
Fig. 3. Mapping the residues that are critical for GRK2 function in Hh signaling.
(A) Domain structure of GRK2 [adapted from (61)]. RH – regulator of G-protein signaling homology; AGC C-tail – C-terminal extension of the kinase domain reminiscent of Protein Kinases A, G, and C; 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-GFP. Each data point represents mean±S.D (n=3). Statistical significance was determined by unpaired Welch’s t-test and depicted as follows: P<0.01 (**).
Fig. 4
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 - floorplate; pMN - motor neuron progenitors; p0, p1, p2, 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 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 sub-types summarized in (A). Each data point represents the data from one image of a NPC colony (see fig. S4A) consisting of 100-200 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 non-parametric ANOVA test and depicted as follows: P<0.0001 (****). (C and D) Immunoblots (representative of two independent experiments) showing the indicated proteins in wild-type (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
Fig. 5. GPR161 suppresses low-level Hh responses and attenuates high-level Hh responses in neural progenitor cells.
(A) Hh signaling was assessed (n=3 independent experiments) using immunoblots of extracts from wild-type and Gpr161-/- NPCs left untreated or treated with SHH. (B) Activation of the GBS-Venus reporter in wild-type (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 GLIR and GLIA 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 wild-type 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 is 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
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 h 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 a NPC colony (see fig. S6A) consisting of 100-200 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 non-parametric ANOVA test and depicted as follows: P<0.0001 (****) and and P>0.05 (ns, not significant). (D) Summary of the effect of GRK2 and SMO inhibitors on the differentiation of wild-type (WT) and Gpr161-/- NPCs.
Fig. 7
Fig. 7. GαS negatively regulates all levels of Hh signaling in NPCs.
(A) Immunoblot (representative of three independent experiments) showing the effect of GRK2 and GRK3 inhibition (by cmpd101) on SHH-induced responses in wild-type (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 (upper panel). 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 (lower panel; 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.

Comment in

  • Grking the Smoothened signal.
    Sharpe HJ, de Sauvage FJ. Sharpe HJ, et al. Sci Signal. 2018 Feb 6;11(516):eaar6377. doi: 10.1126/scisignal.aar6377. Sci Signal. 2018. PMID: 29438011 Review.

Similar articles

See all similar articles

Cited by 15 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback