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The lysosomal GPCR-like protein GPR137B regulates Rag and mTORC1 localization and activity

Abstract

Cell growth is controlled by a lysosomal signalling complex containing Rag small GTPases and mammalian target of rapamycin complex 1 (mTORC1) kinase. Here, we carried out a microscopy-based genome-wide human short interfering RNA screen and discovered a lysosome-localized G protein-coupled receptor (GPCR)-like protein, GPR137B, that interacts with Rag GTPases, increases Rag localization and activity, and thereby regulates mTORC1 translocation and activity. High GPR137B expression can recruit and activate mTORC1 in the absence of amino acids. Furthermore, GPR137B also regulates the dissociation of activated Rag from lysosomes, suggesting that GPR137B controls a cycle of Rag activation and dissociation from lysosomes. GPR137B-knockout cells exhibited defective autophagy and an expanded lysosome compartment, similar to Rag-knockout cells. Like zebrafish RagA mutants, GPR137B-mutant zebrafish had upregulated TFEB target gene expression and an expanded lysosome compartment in microglia. Thus, GPR137B is a GPCR-like lysosomal regulatory protein that controls dynamic Rag and mTORC1 localization and activity as well as lysosome morphology.

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Data availability

The screen data that support the findings of this study have been deposited in the PubChem BioAssay under the accession code AID 29260. Statistical source data for Figs. 28 and Supplementary Figs. 1, 2, 46 and 8 are provided in Supplementary Table 4. All data supporting the findings of this study are available from the corresponding author on request.

Code availability

The MATLAB script for the analysis of lysosomal localization is shared in Supplementary Note.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

K.S. and H.I. were supported by fellowships from A*STAR Singapore and AHA (18POST33990334), respectively. W.S.T. is a Catherine R. Kennedy and Daniel L. Grossman Fellow in Human Biology. Work in the Talbot lab was supported by the NIH grant R01NS050223 and the NMSS grant RG-1707–28694, and in the Meyer lab by R35 GM127026 and UL1TR001085 and the Stanford SPARK Translational Program. We thank D. Garbett, M. Chung, S. Cappell, S. Spencer, S. Collins, M. Koberlin and other lab members for discussions and critical reading of the manuscript, A. Bisaria for plasmids, M. Lopez for the NPC1 construct and D. Solow-Cordero at the HTBC for help in preparing siRNAs for screening.

Author information

K.S. generated the TALEN mutations in zebrafish and analysed microglia. H.I. generated the CRISPR mutations in zebrafish and performed quantitative PCR. K.S., H.I. and W.S.T. designed the zebrafish experiments, analysed the data and contributed to writing the manuscript. L.G. and T.M. planned the initial siRNA screen and follow-up experiments characterizing GPR137B function. T.M. supervised the project and wrote most of the manuscript together with L.G. L.G. and T.M. planned and L.G. performed and analysed all cell-based experiments. A.S., L.G. and T.M. planned and A.S. performed and analysed all biochemistry studies of GPR137B. K.H. and L.G. performed the initial siRNA screen and R.W. helped with the initial analysis of the screen. A.H. helped with imaging. G.D. helped with image analysis and evolutionary analysis of GPR137B. X.G. and J.L. helped with some cell biology and biochemical studies.

Competing interests

The authors declare no competing interests.

Correspondence to Tobias Meyer.

Integrated supplementary information

Supplementary Fig. 1 Additional figure panels describing the primary rpS6 screen and the secondary mTOR translocation screen.

a, rpS6 phosphorylation is a robust and sensitive readout for mTORC1 activation using leucine and various known inhibitors of the mTORC1 pathway. Hs68 cells were starved of serum and amino acids for 2.5 h and restimulated as indicated. Images were quantified by CellProfiler, and the integrated cytoplasmic intensity of phospho-rpS6 was used as a measure of mTORC1 activation. The mean of two independent experiments is shown. b, Screen quality assessed by quantification of siRNA-mediated changes in integrated single-cell rpS6 phosphorylation immunofluorescence intensities for all siControl, siRHEB and siTSC2 wells in the primary screen (n = 384 wells each). Each point represents the average of triplicates for the same well position, normalized by the plate median and the plate s.d. c, The strongest single siRNA effect in each pool of siRNAs dominates the pool behaviour in the primary screen (left). The value of the strongest single siRNA effect is plotted on the x axis against the primary screen value on the y axis. The rpS6 phosphorylation effect of the average of the second and third strongest single siRNA effects is also plotted on the x axis against the primary screen Z-score on the y axis (right). This provides an independent validation that the effect of the siRNA knockdown is specific. d, Pathway analysis (Ingenuity) shows statistical enrichment and the number of deconvolution hits in a subset of canonical pathways (red and green for negative and positive regulators, respectively). Only siRNAs selected as unbiased top hits (988 genes) were used in the analysis. P values were calculated by the right-tailed Fisher’s exact test. e, Image-based correlation analysis shows cycloheximide-stimulated and RagC-dependent colocalization of mTORC1 and Lamp2. Error bars are ±s.d. of the population average; a two-tailed Student’s t-test was used for n = 3 independent experiments.

Supplementary Fig. 2 Validation of GPR137B as a regulator of amino acid-induced mTORC1 translocation to lysosomes.

a, Two independent synthetic siRNAs targeting GPR137B efficiently knockdown GPR137B mRNA levels as measured by quantitative PCR. Error bars are ±s.d. of the population averages; n = 3 independent experiments. b, Knockdown of GPR137B in HeLa cells reduces amino acid-stimulated mTOR translocation. Error bars are ±s.d. of the population average; n = 3 independent experiments and one-way ANOVA followed by Tukey’s test is used. c, Rescue of mTOR translocation by GPR137B expression in HeLa cells treated with 3′ UTR GPR137B siRNA. Error bars ±s.d. of the mean for n = 3 independent experiments and a two-tailed Student’s t-test was used. d, Cells treated with siGPR137B reduces phosphorylation of rpS6 at 240/244 in full media and 120 min after amino-acid stimulation. Hs68 cells treated with control, GPR137B or RAGC siRNAs were left in full media or were amino acid starved and restimulated with amino acids for the indicated time points. n = 3 independent experiments; error bars are mean ± s.d., ratio paired, two-tailed t-test was used. e, GPR137B-mediated increase in mTORC1 translocation is not a result of a change in lysosome size as indicated by the amount of Lamp2 staining. Error bars are ±s.d. from the mean and a paired, two-tailed t-test was used for n = 3 independent experiments. f, Expression of GPR137 and GPR137C mediates an increase in mTOR recruitment in amino acid-starved cells. HeLa cells transfected with GPR137-YFP or GPR137C-YFP were amino acid starved, fixed and stained for GFP and mTOR. The red asterisk indicates transfected cells. Scale bars, 10 μm. g, Quantification of f from n = 3 independent experiments. Error bars are ±s.d. of the mean; a two-tailed Student’s t-test was used. h, Control experiment showing that Lamp1-GFP overexpression does not increase the amino acid-sensitive and Torin-sensitive 4E-BP1 phosphorylation at residues 36/47. Error bars are ±s.e.m. of the population average; P values are calculated by comparing transfected (n = 1,495 cells for no aa, n = 1,629 cells for plus aa and n = 1,577 cells for Torin) to untransfected cells (n = 5,137 cells for no aa, n = 5,499 cells for plus aa and n = 5,031 cells for Torin) in the same well using a two-tailed Student’s t-test. i,j, Lack of rescue of mTOR translocation and activity by GPR137B in serum-starved cells. Hs68 cells starved of serum overnight do not show a statistically significant increase in lysosomal mTOR localization (i) or levels of phosphorylated 4E-BP1 at 37/46 (j). For i and j, n = 3 independent experiments with data representing mean ± s.d.; a two-tailed Student’s t-test was used.

Supplementary Fig. 3 Rag GTPase-dependent mTORC1 translocation and GPR137B–mTOR interaction.

a, Representative images show that mTOR translocation is abolished in Rraga/b−/− MEFs. Scale bars, 20 μm. b, Repeat of mTOR, RagA interaction with GPR137B in MEFs and the requirement of Rag GTPases for the interaction of mTOR and GPR137B. mTOR and RagA co-immunoprecipitation with GPR137B-3×FLAG was examined in control and Rraga/b−/− MEFs stably expressing GPR137B-3×FLAG. Experiments in a and b were performed twice.

Supplementary Fig. 4 Additional experiments evaluating GPR137B interactions and GPR137B as an adaptor of lysosomal RagA/C.

a, Immunoprecipitation of FLAG-NPC1 from lysates of HEK293T cells stably expressing FLAG-tagged NPC1 did not co-immunoprecipitate mTORC1 components or Rag GTPases. Cells are treated as in Fig. 4a. b, GPR137B does not interact with SLC38A9. Co-immunoprecipitation was performed as in Fig. 4a. c, Quantification of Fig. 4c showing the mean of two independent experiments. d, Interaction of GPR137B with mTORC1 and RagA was examined in HeLa cells stably expressing GPR137B-3×FLAG. Experiments in a, b and d were performed twice. e, GPR137B can increase mTORC1 translocation independent of Sestrins. Control (parental HEK293T) cells and SESTRIN1,2,3 triple-null cells transfected with either Lamp1-Turquoise or GPR137B-Turquoise were amino acid starved for 60 min and restimulated for 10 min. GPR137B overexpression still increases mTOR translocation significantly in SESTRIN1,2,3 triple-null HEK293T cells. Data represent mean ± s.d. from four independent experiments and a two-tailed Student’s t-test was used. f, GPR137B regulates RagC lysosomal localization. Increasing the expression of GPR137B causes increased lysosomal RagC localization. Cells were treated and analysed as in Fig. 4d; mean ± s.d. from three independent experiments. GPR137B-expressing bins show statistically significant differences with repeated-measures one-way ANOVA analysis and Tukey’s test. g,h, Lack of rescue of RagA localization and mTORC1 activity by GPR137B expression in siRagulator-treated cells. g, RagA lysosomal localization was quantified in HeLa cells transfected with the indicated siRNA and cDNAs, amino acid starved for 2 h and restimulated for 10 min. All values are normalized to control cells (siControl treated expressing Lamp1). Lamp1-expressing and GPR137B-expressing cells in siLAMTOR3 treated wells in both amino acid-starved and restimulated conditions show similar levels of reduced RagA lysosomal localization. h, Phosphorylation of 4E-BP1 at 37/46 was quantified in cells treated as in g and restimulated with amino acids for 30 min. In siLAMTOR3-treated wells, a paired, two-tailed Student’s t-test shows no statistically significant difference between GPR137B-expressing cells and the untransfected cells of the same well. In g and h, n = 3 independent experiments with data representing mean ± s.d.

Supplementary Fig. 5 Repeat and quantification of Fig. 5 western blots.

a, GPR137B knockdown decreases mTORC1–RagA interactions in amino acid-stimulated cells. HEK293T cells stably expressing HA-RagA was transfected with siGPR137B. HA-RagA was immunoprecipitated and the mTOR and raptor interaction was examined. b, Quantification of a. n = 3. Error bars are ±s.d. of the mean. c, Overexpression of GPR137B increases mTORC1 interactions with RagA. GPR137B-FLAG was overexpressed in HA-RagA-expressing HEK293T cells and HA immunoprecipitation was performed. d, Quantification of c. n = 3. Error bars are ±s.d. of the mean.

Supplementary Fig. 6 GPR137B as an activator of lysosomal RagA/C.

a, GPR137B co-expression with Rap2A does not enable Rap2A expression-dependent recruitment of mTORC1 under amino acid starvation (2 h). Heatmaps show that co-expression of GPR137B-mCherry with Rap2A does not induce Rap2A expression-dependent recruitment of mTORC1 translocation (left), but co-expression of GPR137B with RagA/C causes mTORC1 translocation at low levels of RagA/C expression (right). HeLa cells were amino acid starved for 2 h in dialysed serum, and cells co-expressing GPR137B-mCherry and CFP-Rap2A (n = 958 cells) or GPR137B-mCherry and RagA/CFP-RagC (n = 1,466 cells) were binned into 8 equal bins of CFP intensities and mCherry intensities. The average of the mTOR translocation score for each bin is plotted as a blue-yellow heatmap scaled from 50 to 450. Grey indicates bins for which there was an insufficient number of cells. Heatmaps are plotted from translocation scores pooled from two independent experiments. b, Repeat of experiment in Fig. 5e demonstrating synergistic activation of mTORC1 by GPR137B and wild-type RagA/C. See the Methods section. c, Calibration of wild-type Turquoise-RagC (paired with HA-tagged wild-type RagA) and Q120L CFP-RagC (paired with HA-tagged DN RagA) fluorescence intensity using an endogenous RagA antibody (see Methods). d, Histograms of all single-cell measurements for the expression levels of Lamp1 and GPR137B; the levels of mTOR translocation show dynamic ranges for these three components in Fig. 5e. The red bracket in each histogram shows that most of the values for each parameter is included in the heatmap and therefore represents the population accurately. e,f, Lysosomal Venus-RagC intensity recovers similarly when co-expressed with GPR137B or with Lamp1 in NPRL3/− HEK293E cells in the absence of amino acids after photobleaching (e). Lamp1, n = 9 cells and GPR137B, n = 9 cells. (f) A bar graph representation of data in e is also shown (f). n = 3 independent experiments with data representing mean ± s.d. (e,f); a two-tailed Student’s t-test was used in e.

Supplementary Fig. 7 Validation of C1-domain-tagged RagC constructs in pulling RagA and raptor to the plasma membrane.

a, C1-domain-tagged RagC can pull wild-type RagA to the plasma membrane. The C1 domain fused to N terminus of RagC (C1-RagC) co-expressed with wild-type RagA in HAP1 cells was stimulated with 1 μM PMA and fixed. Scale bars, 10 μm. b, C1-RagC-mediated raptor recruitment to the plasma membrane unlikely involves lysosomes being pulled to the plasma membrane. HEK293T cells co-expressing C1-RagC and YFP-raptor were treated with 1 μM PMA, fixed and stained for Lamp2. C1-RagC colocalization with raptor, C1-RagC colocalization with endogenous Lamp2 and raptor colocalization with Lamp2 are shown in the bottom panels. Scale bar, 10 μm. Experiments were repeated independently two times in a and once in b.

Supplementary Fig. 8 Results of knockouts of GPR137B and of its isoform GPR137 in human cells and of gpr137ba knockout in zebrafish.

a, Relative fold change of mTOR translocation in response to amino acids is not reduced in HAP1 GPR137B-KO and GPR137-KO cells compared to parental control. Mean ± s.d. is plotted for n = 3 independent experiments. b, Table showing the per cent amino acid sequence identity between zebrafish Gpr137 homologues and human GPR137B; Gpr137ba shows the highest sequence similarity to the human GPR137B homologue. c, Amino acid alignment of zebrafish Gpr137ba and human GPR137B showing a high degree of conservation between the two sequences. d, Sequence validation of gpr137ba zebrafish generated by TALEN. e, Wild-type and gpr137ba-mutant zebrafish are viable; images represent whole-fish mounts of the indicated genotypes at 5 dpf (not selected for gender). Scale bar, 50 μm. Images are representative of at least three independent experiments. f, Immunoblot on whole zebrafish tissue shows that gpr137ba/ zebrafish do not have increased LC3B or p62 levels or reduced phosphorylation of S6. Images are representative of three independent experiments. g, The presence and absence of homologues of GPR137, mTOR, Rags and Ragulator in analysed eukaryotic species (n = 179) that have, or do not have, homologues of human GPR137B using a previously described algorithm53. A hand-drawn illustrative human-centric species tree is shown; species that have GPR137B homologues are highlighted. GPR137B homologues are present in all vertebrates examined and in many invertebrates, as well as in eukaryotic species as divergent as D.discoideum and Monosiga brevicollis, but not in more ancestral plants and protists. GPR137B homologues are also not found in fungi, Caenorhabditis elegans and Drosophila model systems.

Supplementary Fig. 9 Unprocessed images of all gels and blots.

Unprocessed western blots in the main figures. Some blots were cut into several pieces and incubated with different antibodies. Left, siGPR137B reduces mTORC1 signalling. Unprocessed blot of Fig. 2l. Right, overexpression of GPR137B desensitizes cells to amino acid withdrawal. Unprocessed blot of Fig. 2m. Co-immunoprecipitation of GPR137B and mTOR regulators. Unprocessed blot of Fig. 4a. Rag GTPases are required for self-interaction of GPR137B. Unprocessed blot of Fig. 4b. Overexpression of Rag GTPase mutants and GPR137B–GPR137B interaction. Unprocessed blot of Fig. 4c. Knockdown of GPR137B reduces amino acid-induced RagA–mTOR interaction. Unprocessed blot of Fig. 5a. GPR137B overexpression increases mTORC1 interaction with RagA. Unprocessed blot of Fig. 5b.

Supplementary information

Supplementary Information

Supplementary Figures 1–9, Supplementary Table and Supplementary Note titles/legend

Reporting Summary

Supplementary Table 1

Excel spreadsheet with all 21,041 human siRNA pools tested in the genome-wide screen

Supplementary Table 2

List of canonical pathways identified by Ingenuity pathway analysis of validated siRNAs

Supplementary Table 3

Ranked list of 15 candidate regulators that reduce cycloheximide-induced mTOR translocation confirmed by at least 2 independent siRNAs

Supplementary Table 4

Statistical source data.

Supplementary Table 5

Antibody list.

Supplementary Note

Matlab script for analyzing lysosomal localization of mTOR and other proteins.

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Fig. 1: A genome-wide siRNA screen in human primary fibroblasts identifies candidate regulators of amino acid-stimulated mTORC1 translocation and activation.
Fig. 2: GPR137B regulates mTORC1 translocation to lysosomes and mTORC1 activation.
Fig. 3: GPR137B regulates mTORC1 through RagA/B.
Fig. 4: GPR137B forms an amino acid-sensitive complex with mTORC1 through Rag GTPases and binds to RagA constitutively as an adaptor for lysosomal localization.
Fig. 5: Evidence that GPR137B can activate RagA/C even in the absence of amino acids.
Fig. 6: Evaluation of the role of GPR137B in regulating Rags by monitoring changes in the exchange rate of RagA/C from lysosomes.
Fig. 7: Active Rag and raptor can rapidly diffuse in the cytoplasm while being in a complex.
Fig. 8: Knockout of human GPR137B and gpr137ba zebrafish mutants show defects similar to Rag pathway mutants.
Supplementary Fig. 1: Additional figure panels describing the primary rpS6 screen and the secondary mTOR translocation screen.
Supplementary Fig. 2: Validation of GPR137B as a regulator of amino acid-induced mTORC1 translocation to lysosomes.
Supplementary Fig. 3: Rag GTPase-dependent mTORC1 translocation and GPR137B–mTOR interaction.
Supplementary Fig. 4: Additional experiments evaluating GPR137B interactions and GPR137B as an adaptor of lysosomal RagA/C.
Supplementary Fig. 5: Repeat and quantification of Fig. 5 western blots.
Supplementary Fig. 6: GPR137B as an activator of lysosomal RagA/C.
Supplementary Fig. 7: Validation of C1-domain-tagged RagC constructs in pulling RagA and raptor to the plasma membrane.
Supplementary Fig. 8: Results of knockouts of GPR137B and of its isoform GPR137 in human cells and of gpr137ba knockout in zebrafish.
Supplementary Fig. 9: Unprocessed images of all gels and blots.