Adgra2^ouchless mislocalizes to the endoplasmic reticulum. (A) Schematic representation of Adgra2, Adgra2^ouchless and Adgra2^ΔLRR3 topology and domain organization. Adgra2^ouchless and Adgra2^ΔLRR3 lack the third LRR motif (red rectangle). The positions of the residue variations resulting from naturally occurring SNPs in adgra2^ouchless are designated by red asterisks. (B) Maximal intensity projection of a confocal z-stack of a WT Tg(kdrl:ras-mCherry) embryo at 36 hpf in lateral view. The red and yellow boxes define, respectively, the magnified areas of the hindbrain vasculature shown in C and the intersegmental vessels shown in E. Scale bar: 100 µm. (C) Maximal intensity projection of a confocal z-stack of WT and adgra2^s984/984 Tg(kdrl:ras-mCherry) embryos at 36 hpf in lateral view after injection of 100 pg of adgra2, adgra2-EGFP, adgra2^ouchless, adgra2^ouchless-EGFP, adgra2^ΔLRR3 or adgra2^ΔLRR3-EGFP mRNA at the one-cell stage. The red arrowheads point to the CtAs invading the hindbrain rhombomeres. Scale bar: 50 µm. (D) Single-plane confocal scans through enveloping layer cells of 5 hpf blastulas injected at the one-cell stage with 50 pg of lyn-RFP mRNA together with 100 pg of adgra2-EGFP, adgra2^ouchless-EGFP or adgra2^ΔLRR3-EGFP mRNA. Scale bar: 50 µm. (E) Single-plane confocal scans through the trunk intersegmental vessels of 30 hpf double-transgenic Tg(kdrl:ras-mCherry); Tg(fliep:Gal4FF) embryos injected at the one-cell stage with 25 pg of Tol2 transposase mRNA and 25 pg of the pTol2-5xUAS:adgra2-EGFP, pTol2-5xUAS:adgra2^ouchless-EGFP and pTol2-5xUAS:adgra2^ΔLRR3-EGFP constructs. Boxes define magnified views of the tip cells presented in the column on the right. Scale bar: 50 µm. (F) Single-plane direct fluorescence confocal scans of non-permeabilized HEK293T cells 48 h after transfection with GPI-RFP, mCherry-SEC61β, Adgra2-EGFP, Adgra2^ouchless-EGFP or Adgra2^ΔLRR3-EGFP encoding constructs. Cells were additionally transfected with reck and Wnt7a (mouse gene) expression constructs. Nuclei were counterstained with Hoechst. Scale bar: 10 μm.

LRR/CT-dependent Adgra2 intracellular trafficking. (A) Schematic representation of Adgra2, Adgra2^ΔLRR1, Adgra2^ΔLRR2, Adgra2^ΔLRR4, Adgra2^ΔLRR, Adgra2^ΔIg-like, Adgra2^ΔHRM, Adgra2^ΔGAIN, Adgra2^ΔLRR/CT, Adgra2^{ΔLRR/CT/Ig-like} and Adgra2^{ΔLRR/CT/Ig-Like/HRM} domain organization. See Fig. 1A for schematic labels. (B) Single-plane direct fluorescence confocal scans of non-permeabilized HEK293T cells 48 h after transfection with the indicated adgra2 variants together with the GPI-RFP membrane marker or the mCherry-SEC61β ER marker. Cells were additionally transfected with reck and Wnt7a (mouse gene) expression constructs. Nuclei were counterstained with Hoechst. Scale bar: 10 μm. (C) Colocalization assessment of Adgra2 and its variants with the membrane marker GPI-RFP (red dots) or the ER marker mCherry-SEC61β (blue dots) using the Pearson correlation coefficient. Error bars represent median±interquartile range. (D) Quantification of neurog1:EGFP⁺ DRG at 72 hpf (red dots) and hindbrain CtAs at 60 hpf (blue dots) in WT and adgra2 morphant larvae and embryos injected at the one-cell stage with 100 pg RNA encoding Adgra2 or Adgra2 variants. Error bars represent median±interquartile range (***P<0.001; ****P<0.0001; Kruskal–Wallis test).

Cellular distribution of Adgra2 LRR/CT domain hybrids. (A) Schematic representation of chimeric Adgra2 receptors in which Adgra2 LRR3 is substituted with the LRR7 (blue hashed rectangle) of CPN2 (CPN2^LRR7), the LRR1 of Adgra2 (Adgra2^LRR1), the LRR2 of Adgra2 (Adgra2^LRR2) or the LRR3 (red hashed rectangle) of Adgra3 (Adgra3^LRR3). See Fig. 1A for schematic labels. (B) Sequence alignment of the LRR motifs illustrated in A. Identical amino acids are highlighted in gray. (C) Single-plane direct fluorescence confocal scans of non-permeabilized HEK293T cells 48 h after transfection with GPI-RFP, mCherry-SEC61β and Adgra2 hybrid-encoding constructs. Cells were additionally transfected with reck and Wnt7a (mouse gene) expression constructs. Nuclei were counterstained with Hoechst. Scale bar: 10 μm. (D) Colocalization assessment of Adgra2 and its variants with the membrane marker GPI-RFP (red dots) or the ER marker mCherry-SEC61β (blue dots) using the Pearson correlation coefficient. Error bars represent median±interquartile range. (E) Quantification of neurog1:EGFP⁺ DRG at 72 hpf (red dots) and hindbrain CtAs at 60 hpf (blue dots) in WT and adgra2 morphant larvae and embryos injected at the one-cell stage with 100 pg of adgra2 or adgra2 hybrid mRNA. Error bars represent median±interquartile range (***P<0.001; ****P<0.0001; Kruskal–Wallis test).

Independent trafficking of Reck and Adgra2 to the plasma membrane. (A) Single-plane confocal images of non-permeabilized HEK293T cells 48 h after transfection with HA-reck and adgra2-EGFP variants, as indicated. (B) Schematic representation of the genetic lesions of ADGRA2^−/− and RECK^−/− cells. The position of the frame-shift mutation is indicated by the red line. See Fig. 1A for schematic labels. (C,D) Single-plane confocal images of non-permeabilized ADGRA2^−/− and RECK^−/− HEK293T cells 48 h after transfection with adgra2-EGFP (C) and HA-reck (D) constructs. In all panels, EGFP is detected by direct fluorescence and the HA-Reck fusion by anti-HA indirect immunofluorescence. Cells were additionally transfected with a Wnt7a (mouse gene) expression construct. Nuclei were counterstained with Hoechst. Scale bars: 10 μm.

Cellular distribution of Adgra2 and Reck interaction. (A) Single-plane confocal images of saponin-permeabilized HEK293T cells 48 h after transfection with HA-reck and adgra2-EGFP variants, as indicated. Nuclei were counterstained with DAPI. EGFP is detected by direct fluorescence and the HA-Reck fusion by anti-HA indirect immunofluorescence. Arrows point to the ER. (B) Proximity ligation assays in HEK293T cells 48 h after transfection with FLAG-adgra2, FLAG-adgra2^ouchless and HA-reck constructs, as indicated. Nuclei were counterstained with DAPI. In all panels, cells were additionally transfected with a Wnt7a (mouse gene) expression construct. Scale bar: 10 μm.