Quantitative Evaluation of Receptor-Mediated Endocytosis

This chapter describes assays, experimental procedures, and analyses that are designed to allow a quantitative evaluation of the process of receptor-mediated endocytosis. It emphasizes on both a theoretical and a practical approach to these issues. Receptors generally represent only a very small fraction of the cell's constituent molecules. Therefore, the analysis of ligand-receptor binding and endocytosis almost invariably requires sensitive assays for ligand–receptor interaction, generally achieved by incorporation of radioactive atoms into ligands. Radiolabeling potentially can compromise important ligand parameters—including bioactivity, specificity, and intracellular trafficking. The entire cell complement of receptors for a specific ligand is composed of several distinct but overlapping subpopulations. These populations differ with respect to their location, their functional capacity for binding ligand, and their capability for recycling. Quantitative evaluation of unoccupied functional receptors involves determining the amount of ligand–receptor complex formed as a function both of time and of concentration of ligand added. The Scatchard approach for determining ligand–receptor binding parameters has been used in characterizing the asialoglycoprotein receptor (ASGP-R) on Hep G2 cells.