Quantitative Analysis of Ligand-Induced Supramolecular Clustering of Death Receptor 5 in Jurkat Cells

Abstract

Death receptor 5 is a transmembrane protein belonging to the tumor necrosis factor receptor superfamily. Upon ligand binding, DR5 and several members of its superfamily form supramolecular clusters, often modeled as highly organized lattices wherein receptors are retained in their pre-ligand oligomeric assemblies and crosslinked by ligand into high molecular weight networks. Networks can be visualized by fluorescent confocal microscopy, however there is some disagreement as to whether these punctate features are in fact protein assemblies, membrane microdomains (lipid rafts), or a combination of both. Our data show that while ligand induced DR5 networks do assemble in both the presence and absence of lipid rafts, key differences exist in their ability to initiate apoptotic signaling and in the oligomeric structure of the receptor as it is subsumed within the ligand-receptor network. To identify subtle structural differences between supramolecular networks in the presence and absence of lipid rafts, we have developed a technique to detect individual networks and quantitatively compare them between treatment groups in terms of total ligand bound, relative size, and density.