Metabolism and Systems Science and Centre for Diabetes, Endocrinology and Metabolism (CEDAM), University of Birmingham, Birmingham, UK.
Centre for Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, Birmingham, UK.
Methods Mol Biol. 2025;2861:111-126. doi: 10.1007/978-1-0716-4164-4_9.
The calcium-sensing receptor (CaSR), which regulates parathyroid hormone secretion by sensing serum calcium concentrations, has developed unique trafficking mechanisms to respond to constant exposure to its orthosteric ligand calcium. CaSR rapidly responds to fluctuations in serum calcium by driving forward trafficking of the receptor to cell surfaces in a mechanism known as agonist-driven insertional signaling (ADIS). This increase in CaSR at cell surfaces is counterbalanced by both constitutive and agonist-driven internalization of the receptor. Deciphering these mechanisms is important to understand how mutations in the CaSR and components of its signaling and trafficking pathways cause human disorders of calcium homeostasis.This chapter describes a protocol to measure CaSR ADIS and endocytosis in parallel using total internal reflection fluorescence (TIRF) microscopy. This utilizes a mammalian expression construct comprising the full-length human CaSR with an N-terminal bungarotoxin minimal-binding site that can be labeled with commercially available fluorescent ligands to measure endocytosis, and a super-ecliptic pHluorin (SEP) to measure total cell surface expression and exocytic events. This protocol could easily be adapted to simultaneously assess forward trafficking and endocytosis of other membrane proteins by TIRF microscopy.
钙敏感受体（CaSR）通过感应血清钙浓度来调节甲状旁腺激素的分泌，它已经发展出独特的运输机制来应对其正位配体钙的持续暴露。CaSR 通过正向运输受体到细胞表面的机制（称为激动剂驱动的插入信号（ADIS））快速响应血清钙的波动。这种细胞表面 CaSR 的增加被受体的组成型和激动剂驱动的内化所平衡。解析这些机制对于理解 CaSR 及其信号和运输途径的突变如何导致人类钙稳态紊乱至关重要。本章描述了一种使用全内反射荧光（TIRF）显微镜平行测量 CaSR ADIS 和内吞作用的方案。该方案利用哺乳动物表达构建体，其中包含全长人 CaSR，其 N 端带有一个最小结合部位的箭毒毒素，该部位可以用市售的荧光配体标记，以测量内吞作用，并用超亮型 pH 荧光素（SEP）测量总细胞表面表达和外排事件。该方案可以通过 TIRF 显微镜轻松地适应同时评估其他膜蛋白的正向运输和内吞作用。
