Modulation mechanism of L-tryptophan and phosphate to the chicken extracellular calcium-sensing receptor via downstream G protein-coupled signaling cascades and their binding modes

Laying hens undergo dynamic bone turnover and have a high demand for calcium due to the daily egg-laying cycle. The chicken extracellular calcium-sensing receptor (cCaSR) plays an important role in calcium homeostasis. This study investigated the modulatory effects and mechanisms of L-tryptophan (L-Trp) and inorganic phosphate (PO₄³⁻) on cCaSR-mediated signaling. A stable Flp-In™-293 cell line expressing cCaSR was established and used in a completely randomized in vitro cell culture design. Individual cell culture wells served as the experimental unit, with at least three technical replicates per experiment and at least three independent experiments. cCaSR activation was evaluated using luciferase reporter assays and a cAMP assay to assess Gαq-, Gβγ/Gαi-, Gα12-, and Gαi-mediated signaling. Dose-response curves were fitted by nonlinear regression, and data were analyzed by one-way ANOVA with Dunnett’s or Tukey’s multiple-comparison test (P < 0.05). L-Trp enhanced Ca²⁺-mediated cCaSR activation, shifting the Ca²⁺ EC50 by 12.88%, 5.18%, and 1.02% in the Gαq, Gβγ/Gαi, and Gα12 pathways, respectively, whereas the Ca²⁺ IC50 for cAMP shifted by 10.15% in the Gαi pathway. The calcimimetic R568 was used to partially substitute for Ca²⁺ to minimize Ca²⁺-phosphate interactions. In the presence of Ca²⁺ and R568, 2 mM PO₄³⁻ inhibited cCaSR responses in the Gαq, Gβγ/Gαi, and Gα12 pathways by 92.97%, 31.50%, and 30.23%, respectively, compared with 0.9 mM PO₄³⁻ (P < 0.05). At 5 mM PO₄³⁻, cAMP levels increased by 51.20% (P < 0.05). The greatest numerical changes induced by both L-Trp and PO₄³⁻ were observed in the Gαq-mediated NFAT-RE pathway. Molecular docking and dynamics simulations predicted one L-Trp binding site and two PO₄³⁻ binding sites in the extracellular domain of cCaSR, with hydrogen bonding contributing to both interactions and hydrophobic interactions additionally contributing to L-Trp binding. These findings demonstrate that L-Trp and PO₄³⁻ act as positive and negative allosteric modulators, respectively, with pathway-dependent effects on cCaSR signaling, providing mechanistic evidence supporting cCaSR as a potential nutritional target for further research on calcium homeostasis and bone health in laying hens.