Discrete Signaling Regions in the Lymphotoxin-β Receptor for Tumor Necrosis Factor Receptor-associated Factor Binding, Subcellular Localization, and Activation of Cell Death and NF-κB Pathways

Abstract Lymphotoxin-β receptor (LTβR), a member of the tumor necrosis factor receptor superfamily, is essential for the development and organization of secondary lymphoid tissue. Wild type and mutant LTβR containing successive truncations of the cytoplasmic domain were investigated by retrovirus-mediated gene transfer into HT29.14s and in 293T cells by transfection. Wild type receptors accumulated in perinuclear compartments and enhanced responsiveness to ligand-induced cell death and ligand-independent activation of NFκB p50 dimers. Coimmunoprecipitation and confocal microscopy mapped the TRAF3 binding site to amino acids PEEGDPG at position 389. However, LTβR truncated at position Pro379 acted as a dominant positive mutant that down-modulated surface expression and recruited TRAF3 to endogenous LTβR. This mutant exhibited ligand-independent cell death and activated NF-κB p50 dimers. By contrast, truncation at Gly359 created a dominant-negative mutant that inhibited ligand-induced cell death and activation of NF-κB p50/p65 heterodimers. This mutant also blocked accumulation of wild type receptor into perinuclear compartments, suggesting subcellular localization may be crucial for signal transduction. A cryptic TRAF-independent NF-κB activating region was identified. These mutants define discrete subregions of a novel proline-rich domain that is required for subcellular localization and signal transduction by the LTβR.