Fig. 4: Differential analysis of the cGAL53 and cGAL29 signaling pathways.

HEK293 cells were transfected with 2 μg of GALR plasmids and 50 ng of a reporter gene plasmid. Peptides were added 36 h post-transfection, and luciferase activity was measured 12 h after stimulation. a–c G protein pathway activation of GALR2 after treatment with cGAL53 and cGAL29, where cGALR2, hGALR2, and mGALR2 represent chicken, human, and mouse GALR2. d–f G protein pathway activation of GALR3 in response to cGAL53 and cGAL29 treatment, as assessed by luciferase assay technical triplicate. g β-arrestin2 recruitment by GALR2 following cGAL53 and cGAL29 treatment, measured via the Tango assay. h β-arrestin2 recruitment by GALR3 after cGAL53 and cGAL29 treatment, using the Tango assay. i β-arrestin2 pathway activation of GALR2, measured via BRET assay. j Gq activation of GALR2 measured via BRET assay. Data are represented as mean ± SD. Calcium responses were measured by fluorescence intensity in GALR2 (k) and GALR3 (l) overexpressing cells following stimulation with various concentrations of GAL peptides. ATP (10 mM) and HBSS alone were used as positive and negative controls, respectively. m Schematic diagram of cGAL53 peptide truncations; note that in addition to the insert, alternative splicing leads to a different amino acid (Tyr versus His) just beyond the insertion. n Effects of cGAL53 truncations on the G protein pathway and β-arrestin2 recruitment to GALR2. Data in (a–j, n) are presented as mean ± SD from three independent experiments; data in (k, l) are presented as mean values from three independent experiments only.