Fig. 3

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).