Visualization and Characterization of Receptor Clusters by Transmission Electron Microscopy

Abstract

Publisher Summary Multivalent ligands often bind avidly to their target receptors. In addition, they can simultaneously bind multiple receptors and cause them to become clustered. Receptor clustering by multivalent ligands modulates the cell adhesion and signaling functions of many carbohydratebinding proteins, including galectins and selectins. Not all clusters, however, are equivalent in signaling potency or adhesion properties. Clusters that contain multiple copies of a receptor often elicit greater responses; therefore, the stoichiometry of receptor–ligand clusters can be an important determinant of activity. The prevalence of multivalency in protein–carbohydrate interactions mandates that new methods to characterize multivalent binding should be developed. Although many assays focus on assessing the apparent affinity of a multivalent carbohydrate–lectin interaction, many multivalent ligands promote lectin clustering. Because the clustering of a lectin can interfere with or augment its function, methods to characterize lectin–ligand assemblies are needed. The TEM method described in this chapter facilitates the visualization and characterization of such clusters. In addition, using this technique, the effects of ligand valency on cluster size can be investigated. Another potential use of this TEM method is in exploring heterogeneous multireceptor clusters.