Membrane receptor internalization represents one of the kay MOAs of many drug compounds. Receptor internalization, or endocytosis, is a part of the signal transduction of cells. It plays an essential part in diverse physiological processes, e.g. cancer, virology, neurotransmitters, antibody-drug conjugates (ADC) development. Creative Biolabs presents comprehensive assay services for compound in vitro function validations. Currently, we provide professional compound internalization assessment service based on fluoregen activating protein (FAP) technique, which has been widely applied for measuring receptor internalization, trafficking, ligand selectivity and receptor re-sensitization.
Background of Receptor Internalization
Receptor-mediated endocytosis is an endocytotic process in which specific molecules are taken into the cell when binding to surface receptors. It is highly receptor-specific, and not all surface receptors are capable of triggering ligand internalization. Upon ligand binding, the ligand-receptor complex will diffuse through the plasma membrane until captured by a preformed or forming clathrin-coated pit. Some receptors can nucleate a clathrin-coated pit around the receptor. A mature pit will break away from the plasma membrane and from a clathrin-coated vesicle which then fusing to an early endosome. Once fused the endocytosed cargo (receptor and/or ligand) can then be sorted to lysosomal, recycling, or other trafficking pathways.
Fig. 1 Mechanism of clathrin-dependent endocytosis.1
Magic™ Fluorogen Activating Protein Technology
Fluorogen activating peptids (FAPs) are special small reporters that emit fluorescence only in presence of a non-fluorescent molecule (fluorogen). FAPs can be fused to the exterior (or interior) face of the membrane protein of interest, while a standard fluorescent protein of different color (EGFP or mRFP) can then be fused on the interior (or exterior) face. Since the fluorogen cannot penetrate cells, only receptors that are at the cell surface or those that visited the cell surface will be visible. Based on this FAP-fluorogen pair, cell lines engineered with FAP/fluorescent tag can be an effective tool to assay compounds with internalization potencies. As shown in Fig. 2, after incubation with certain compound, receptors on cell surface & in cytosol can be obviously distinguished and quantified.
Fig. 2 Fluorescence images of dumbbell constructs in NIH 3T3 cells.2
The leftmost column is the diagram of the topology of each fusion protein on the lipid bilayer, where the top refers to the extracellular and the bottom refers to the intracellular. The next three columns represent green, red and merge fluorescence channels respectively. The first line of images is acquired with a cell expressing a membrane protein with a FAP tag (green) on the N-terminus (outside of the cell) and RFP on the C-terminus (inside of the cell). The FAP image (A) only shows membrane protein that was synthesized and transport to the cell surface using membrane-impermeant fluorogen whereas the RFP image (B) shows all tagged protein regardless of location. The second lines of images show the results if a red FAP and GFP are used.
Featured Advantages of FAP/Fluorogen Technique
Scientists in Creative Biolabs are pleased to leverage our expertise and technologies to assist our clients with their cutting-edge projects. Currently, we offer a full range of ready-to-use FAP-tagged cell lines to assay compounds and characterize their internalization activities. We are also happy to establish custom cell line upon request. Please contact us for more information and a detailed quote.
References
For Research Use Only.
