G Protein-Coupled Receptor 37L1 Modulates Epigenetic Changes in Human Renal Proximal Tubule Cells

Figure 1. Glycerol gradient fraction of GPR37L1 protein complex. Protein preparations from mock- or GPR37L1-GFP-transfected hRPTCs were subjected to glycerol gradient (10–30%) centrifugation. (A) Fractions collected from the top of the gradient were subjected to immunoblotting for GPR37L1-GFP. Positions of the molecular size markers are indicated on the left. Positions of GPR37L1-GFP * monomer and ** high molecular aggregate are indicated on the right. (B) Graph showing the densitometric quantification of GPR37L1-GFP in the glycerol gradient fractions (blue). Fractions 10–14 (red) containing the highest levels of GPR37L1-GFP were pooled and used for co-immunoprecipitation (Co-IP), followed by mass spectrometry analyses.

Figure 2. GPR37L1-GFP localized at the plasma and the nuclear membrane. Immunofluorescence images of whole cell and nuclei of hRPTCs showing the expression of GPR37L1-GFP at the plasma and the nuclear membrane. hRPTCs grown on glass bottom microwell dishes were transfected with the plasmid GPR37L1-GFP. Two days following transfection, the (A) whole cells or (B) nuclei prepared as described in the methods were fixed with 1% PFA for 10 min. Subcellular locations of GPR37L1-GFP expression were detected by fluorescence confocal microscopy at 600× magnification. GPR37L1-GFP, green; nucleus, blue. An example for the locations of the GPR37L1-GFP on the plasma membrane and nuclear membrane are indicated by the white and red arrows, respectively.

Figure 3. GPR37L1 protein has a nuclear localization signal. Nuclear localization signal (NLS) and immunoblots showing the expression of GPR37L1 in the nucleus. (A) Amino acid sequence of GPR37L1 showing the potential bipartite (underlined) and classical monopartite (bold) NLS sequence on the extracellular domain amino terminus, predicted using the nuclear localization signal predictor tools http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi (accessed on 15 March 2019) and http://mleg.cse.sc.edu/seqNLS/ (accessed on 15 March 2019). The signal peptide sequence, the first 25-amino-acid sequence at the amino terminus, is indicated in italics. Extracellular domains are indicated in blue, transmembrane helices in red, and intracellular domains in black. (B–E) hRPTCs were transfected with a plasmid encoding GPR37L1-GFP or with a plasmid pcDNA, an empty vector (mock). (B) Two days after transfection, the expression of GPR37L1-GFP in total (T) or nuclear (N) protein preparations was detected by immunoblotting using an antibody specific to GFP. The same blot was probed sequentially for (C) calnexin, a marker for endoplasmic reticulum; (D) Na+/K+-ATPase, a marker for membrane proteins; or…

Figure 4. NLS for GPR37L1 is located on the amino terminus. (A) N-terminus (N-ter) 124-amino-acid sequence of GPR37L1 showing the potential monopartite (underlined) NLS sequence, NLS wild-type, and NLS mutant sequences fused to luciferase–GFP fusion (LG) constructs. (B) Diagram showing the plasmid constructs of wild-type or the N-terminus mutants of GPR37L1 tagged to LG fusion cDNA (LG) (37L1-Nter-LG) or the wild-type or mutant NLS tagged to LG (37L1-WtNLS-LG or 37L1-MuNLS-LG). (C) hRPTCs were transfected with the indicated plasmids. The wild-type or mutant GPR37L1 expressions were determined by immunoblotting of the total or nuclear protein preparations, using two-color infrared fluorescent protein detection. Expression of the LG constructs was detected using the GFP antibodies (green). The blot was probed sequentially for HDAC2 (red), a marker for nuclear protein, or GAPDH (red), a marker for cytoplasmic proteins. IRDye® 800CW-labeled donkey anti-rabbit IgG (926-32213, Licor, Lincoln, NE, USA) was used for GPR37L1-GFP, and IRDye® 680RD-labeled donkey anti-mouse IgG (926-68022, Licor, Lincoln, NE, USA) was used for HDAC2 and GAPDH. hRPTCs transfected with pcDNA, an empty vector, served as…

Figure 5. Amino terminus NLS is required for nuclear expression of GPR37L1. hRPTCs were transfected with the indicated GPR37L1 constructs as in Figure 4A. Two days later, the expressions of wild-type or mutant GPR37L1 proteins were examined by immunofluorescence confocal microscopy at 600× magnification. The cells were also stained for lamin B1, a marker for nuclear inner membrane, and DAPI, a marker for nucleus. GPR37L1, green; nucleus, blue; lamin B1, red; plasma membrane, purple. White dots in the merge column indicate the expression of WtNLS-LG at the nuclear envelope.

Figure 6. GPR37L1 interacts with DNMT1, mTOR, and PRKDC. The glycerol gradient fractions 10–14 of the protein preparation from hRPTCs expressing GPR37L1-GFP (37L1-GFP) or the mock-transfected hRPTCs (mock) from (Figure 1) were used for Co-IP using the GFP antibody (GFP) or control rabbit IgG (IgG). The Co-IP samples were immunoblotted to detect GPR37L1-GFP, DNMT1, mTOR, or PRKDC. The molecular size markers are indicated on the right.

Figure 7. GPR37L1 activates PI3/AKT/mTOR signaling. PI3K/AKT/mTOR signaling components in hRPTCs expressing GPR37L1 were probed by immunoblotting. The hRPTCs were transfected with GPR37L1 (GPR37L1) or control plasmid (pcDNA). (A) Immunoblots of GPR37L1 in hRPTCs transfected with pGPR37L1. (B–D) Quantification of phospho-ERK1/2,(p-ERK1/2), phospho-PI3K (pPI3K), and phospho-AKT (S473) (pAKT) was normalized to the corresponding total (t) protein. n = 3/group, * p < 0.05 vs. their respective pcDNA, t-test.

Figure 8. GPR37L1 decreases DNMT1 expression through mTOR. The hRPTCs, transfected with GPR37l1 or control plasmid (pcDNA), were treated with vehicle (Veh) or rapamycin (50 nM [Rap], for 24 h). (A,B) Protein preparations from the hRPTCs were immunoblotted for DNMT1. Quantification of DNMT1 expression normalized by tubulin. n = 3/group, * p < 0.05 vs. pcDNA+Veh, t-test.

Figure 9. Rapamycin treatment increases DNMT1 and NHE3 expression in hRPTCs. Cells were treated with rapamycin at the concentrations of 1, 10, 20, and 50 nM or vehicle for 24 h. The expression of DNMT1 was determined by Western blot. Quantification was normalized by tubulin expression. The molecular size markers are indicated on the left. Another set of hRPTCs were treated for 24 h with 50 nM rapamycin or vehicle, and the expression of NHE3 of the cell lysates was determined by Western blot. Quantification was normalized by GAPDH. The molecular size markers are indicated on the left. n = 3/group * p < 0.05 vs. vehicle.

Figure 10. GPR37L1 inhibits genomic DNA methylation. (A) Genomic DNA prepared form hRPTCs expressing GPR37L1-GFP were analyzed for 5-cytosine methylation using EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA). n = 3/group, * p < 0.05 vs. GPR37L1/Veh, t-test. (B) Methylation status of NHE3 promoter region was determined by methylated DNA immunoprecipitation followed by quantitative PCR. n = 3/group, + p < 0.05 vs. 5mC-Ab/Mock/Veh, t-test. ++ p < 0.05 vs. 5mC-Ab/GPR37L1/Veh, t-test.

Figure 11. Schematic diagram showing the GPR37L1 signaling pathway in hRPTCs. In hRPTCs, GPR37L1 located on the nuclear membrane signals via the Gβγ to phosphorylate ERK/1/2, which in turn phosphorylates PI3K and activates AKT, a main downstream target of mTOR. Activation of mTOR inhibits DNMT1 in the nucleus and increases the transcription of NHE3 through a DNMT1-mediated decrease in the methylation of the NHE3 promoter.