GPR124 functions as a WNT7-specific coactivator of canonical β-catenin signaling

Abstract

G protein-coupled receptor 124 (GPR124) is an orphan receptor in the adhesion family of GPCRs, and previous global or endothelial-specific disruption of Gpr124 in mice led to defective CNS angiogenesis and blood-brain barriergenesis. Similar developmental defects were observed following dual deletion of Wnt7a/Wnt7b or deletion of β-catenin in endothelial cells, suggesting a possible relationship between GPR124 and canonical WNT signaling. Here, we show using in vitro reporter assays, mutation analysis, and genetic interaction studies in vivo that GPR124 functions as a WNT7A/WNT7B-specific costimulator of β-catenin signaling in brain endothelium. WNT7-stimulated β-catenin signaling was dependent upon GPR124's intracellular PDZ binding motif and a set of leucine-rich repeats in its extracellular domain. This study reveals a vital role for GPR124 in potentiation of WNT7-induced canonical β-catenin signaling with important implications for understanding and manipulating CNS-specific angiogenesis and blood-brain barrier-genesis.

Figure 1. Gpr124 is required for β-catenin signaling in brain endothelium in vivo

A) β-gal positive nuclei (blue; arrows), indicative of β-catenin signaling, were observed in the isolectin (ISL)-positive vessels (red) that penetrated the lateral ventricular wall in the telencephalon (neopallium) of Gpr124^+/+; BAT-Gal⁺ mice (left panel). Blue nuclei were absent from the glomeruloid vessel structures (yellow arrowheads) found in the lateral ventricular wall of the telencephalon of Gpr124^−/−; BAT-Gal⁺ mice (right panel). Bars: 50 μm. B) Quantification of the number of β-gal positive nuclei per vessel structure in Gpr124^+/+; BAT-Gal⁺ (WT) or Gpr124^−/−; BAT-Gal⁺ (KO) neopallium. * p<0.0001 C) β-gal positive nuclei (blue) were observed in isolectin (ISL)-positive vessels (red) throughout the spinal cord of Gpr124^+/+; BAT-Gal⁺ mice (left panel). Blue nuclei were observed in the dorsal (D) side of Gpr124^−/−; BAT-Gal⁺ spinal cord but were absent from the glomeruloid vessel structures (yellow arrowheads) found in the ventral (V) side (inset, right panel). Bars: 100 μm. D) Quantification of the number of β-gal positive nuclei per vessel structure in the ventral or dorsal region of Gpr124^+/+; BAT-Gal⁺ (WT) or Gpr124^−/−; BAT-Gal⁺ (KO) spinal cord. *p<0.0001. The rare β-gal+ nuclei found in Gpr124^−/− forebrain (B) or in the ventral region of the spinal cord (D) were located in vessel stalks interconnecting the glomeruloids with the underlying PNVP. Data in (B) and (D) are the mean ± SEM

Figure 2. GPR124 is a ligand-specific co-activator of canonical β-catenin signaling

A) Transfection of BKO reporter cells with increasing concentrations of FLAG-GPR124 caused a dose-dependent increase in luciferase activity in response to WNT7A. B) WNT7A and WNT7B with and without a myc tag stimulated luciferase activity in BKO cells in combination with GPR124. *p<0.001. C) Mouse and human GPR124 both enhanced WNT7A induced luciferase activity in BKO cells. D) N-terminal FLAG or C-terminal myc tags did not alter GPR124’s ability to co-activate WNT7A signaling. GPR125 did not enhance WNT7A signaling. p<0.01 vs pcDNA plus WNT7A. E) QPCR analysis revealed higher levels of Axin2 and Apcdd1 mRNA expression in doxycycline induced BKO-124i cells. F) GPR124 selectively enhanced WNT7A and WNT7B induced luciferase activity in BKO-124i cells. *p<0.004 G) GPR124 enhanced the activity of ectopically-expressed WNT7 in BKO-124i cells. HEK293 cells were transfected with pcDNA3.1 (pcDNA), WNT2, WNT7A or WNT7B then 24h later mixed with BKO-124i reporter cells and co-cultured for 24h in the presence or absence of doxycycline (Dox) prior to luciferase analysis. *P=0.04, **p=0.005. Data are the mean ± SEM. See also Figure S2 and S3.

Figure 3. Mutation analysis identifies amino acids of WNT7A and GPR124 important for β-catenin signaling

A) Western blotting reveals that each of the mutant WNT7A proteins was expressed in BKO cells at a level similar to the wildtype (WT) WNT7 protein. The schematic on top depicts the relative location of each mutation as well as the C-terminal myc tag. B) Luciferase activity in BKO cells increased in response to wildtype (WT) WNT7A upon co-expression of GPR124 but was attenuated by each of the WNT7A mutations. *p<0.002 C) Co-immunoprecipitation (IP) studies revealed binding of HA-tagged DLG1 (DLG-HA) to full-length FLAG-tagged GPR124 (GPR124-FL) but not the 4 amino acid deletion (GPR124-Δ4aa). IB: Immunoblotting antibody. D) WNT7A-induced luciferase activity in BKO reporter cells in response to increasing amounts of either GPR124-Δ4aa or the full-length vector (GPR124-FL). A constant amount of WNT7 (40 ng) was transfected into samples marked “+”. E) Immunoblotting with anti-FLAG antibodies revealed similar levels of protein following transient transfection of cells with the full-length FLAG-tagged GPR124 receptor (GPR124-FL) and the corresponding 4 amino acid deletion (GPR124-Δ4aa). βactin was used as a loading control. F) Immunofluorescence staining of non-permeabilized cells with anti-FLAG antibodies (green) revealed similar expression of GPR124-FL (full-length) and GPR124-Δ4aa in transfected BKO cells. The nuclei (blue) were counterstained with DAPI. Bar: 10μm. G) Schematic diagram of full-length (FL) GPR2124 and the N-terminal deletion mutants. SP: signal peptide, 7TM: 7 pass transmembrane domains. H) Incorporation of an RGE motif in place of the RGD site did not alter GPR124’s ability to activate WNT7A β-catenin signaling. N.S: non-significant. I) Western blotting revealed expression of the N-terminal GPR124 deletions. The anti-myc antibody also cross-reacted with a product present in all lanes (asterisk) providing a convenient internal loading control. J) Immunofluorescence staining of permeabilized BKO cells with anti-myc antibodies (green) 48h post-transfection revealed a similar membrane staining pattern for GPR124-myc (full-length GPR124 with a C-terminal myc tag) and each of the N-terminal deletions. The nuclei (blue) were counterstained with DAPI. Bar: 10μm. K) BKO reporter assays revealed a loss of β-catenin stimulation by all of the N-terminal GPR124 deletions. *p=0.0001 L) BKO reporter assays revealed that the LRR domain of GPR125 is able to partially rescue luciferase activity in the ΔLRR mutant. Data are the mean ± SEM.

Figure 4. Genetic interaction studies reveal co-operation between Gpr124, Wnt7a and Wnt7b in vivo

A) Isolectin (ISL) staining revealed unusual glomeruloid structures (arrowheads) in the ganglionic eminence (GE) of Gpr124^+/−; Wnt7a^−/−; Wnt7b^+/− forebrains. Note the reduced level of Glut1 in the glomeruloid structures. V: ventricle. Bar: 100μm. B) Isolectin (ISL) staining revealed unusual glomeruloid structures (arrows) in the ventral (v) region of Gpr124^+/−; Wnt7a^−/−; Wnt7b^+/− spinal cords. Bar: 100μm. See also Figure S4.