Protein-Labeling Fluorescent Probe for Folate Receptor α.

GPI-anchored folate receptor α (FRα) is an attractive anticancer drug target and diagnosis marker in fundamental biology and medical research due to its significant expression on many cancer cells. Currently, analyses of FRα expression levels are usually achieved using immunological methods. Due to the continual FRα synthesis and degradation, immunological methods are not suitable for studying real-time dynamic activities of FRα in living cells. In this paper, we introduce a rapid and specific FRα protein-labeling fluorescent probe, FR1, to facilitate the study of the dynamics of expression and degradation processes of endogenous FRα in living cells. With this labeling probe, insights on FRα protein lifetime and shedding from the cell surface can be obtained using fluorescence live-cell imaging and electrophoresis techniques. We revealed that FRα undergoes soluble domain release and endocytosis degradation simultaneously. Imaging results showed that most of the membrane FRα are transported to the lysosomes after 2 h of incubation. Furthermore, we also showed that the secretion of a FRα soluble domain into the environment is most likely accomplished by phospholipase. We believe that this protein-labeling approach can be an important tool for analyzing various dynamic processes involving FRα.

Protein-Labeling Fluorescent Probe Reveals Ectodomain Shedding of Transmembrane Carbonic Anhydrases.

Ectodomain shedding is a form of limited proteolysis in which a protease cleaves a transmembrane protein, releasing the extracellular domain from the cell surface. Cells use this process to regulate a wide variety of biological events. Typically, immunological detection methods are employed for the analysis of ectodomains secreted into the cultured media. In this paper, we describe a new strategy using an affinity-based protein-labeling fluorescent probe to study ectodomain shedding. We analyzed the ectodomain shedding of cell surface carbonic anhydrases (CAIX and CAXII), which are important biomarkers for tumor hypoxia. Using both chemical and genetic approaches, we identified that the ADAM17 metalloprotease is responsible for the shedding of carbonic anhydrases. Compared to current immunological methods, this protein-labeling approach not only detects ectodomain released into the culture media but also allows real-time living cell tracking and quantitative analysis of remnant proteins on the cell surface, thereby providing a more detailed insight into the mechanism of ectodomain shedding as well as protein lifetime on the cell surface.