Ligand-Receptor and Receptor-Receptor Interactions Act in Concert to Activate Signaling in the Drosophila Toll Pathway

Alexander N.R Weber,Martin C Moncrieffe,Monique Gangloff,Jean-Luc Imler,Nicholas J Gay

doi:10.1074/jbc.m502074200

Abstract

In Drosophila, the signaling pathway mediated by the Toll receptor is critical for the establishment of embryonic dorso-ventral pattern and for innate immune responses to bacterial and fungal pathogens. Toll is activated by high affinity binding of the cytokine Spätzle, a dimeric ligand of the cystine knot family. In vertebrates, a related family of Toll-like receptors play a critical role in innate immune responses. Despite the importance of this family of receptors, little is known about the biochemical events that lead to receptor activation and signaling. Here, we show that Spätzle binds to the N-terminal region of Toll and, using biophysical methods, that the binding is complex. The two binding events that cause formation of the cross-linked complex are non-equivalent: the first Toll ectodomain binds Spätzle with an affinity 3-fold higher than the second molecule suggesting that pathway activation involves negative cooperativity. We further show that the Toll ectodomains are able to form low affinity dimers in solution and that juxtamembrane sequences of Toll are critical for the activation or derepression of the pathway. These results, taken together, suggest a mechanism of signal transduction that requires both ligand-receptor and receptor-receptor interactions.

Highlights

In Drosophila, the signaling pathway mediated by the Toll receptor is critical for the establishment of embryonic dorso-ventral pattern and for innate immune responses to bacterial and fungal pathogens
Purified Toll ectodomain was titrated into the Isothermal Titration Calorimetry (ITC) measuring cell containing Spatzle C106 protein (Fig. 1A)
Our ITC data show that two thermodynamically similar modes of binding exist in solution: a 1:1 complex existing when Spatzle C106 is in excess and a 2:1 complex predominating when Toll is in excess

Summary

Introduction

In Drosophila, the signaling pathway mediated by the Toll receptor is critical for the establishment of embryonic dorso-ventral pattern and for innate immune responses to bacterial and fungal pathogens. The two binding events that cause formation of the cross-linked complex are nonequivalent: the first Toll ectodomain binds Spatzle with an affinity 3-fold higher than the second molecule suggesting that pathway activation involves negative cooperativity. We further show that the Toll ectodomains are able to form low affinity dimers in solution and that juxtamembrane sequences of Toll are critical for the activation or derepression of the pathway These results, taken together, suggest a mechanism of signal transduction that requires both ligand-receptor and receptor-receptor interactions. The Toll family of type I transmembrane receptors mediate cellular signal transduction pathways that are central to innate immune responses in both vertebrates and invertebrates [1] These receptors are responsible for detecting microbial molecular patterns, for example bacterial lipoproteins and lipopolysaccharides, and activating a cellular defense response. Structures of the fibroblast growth factor (FGF) receptor in complex with FGF show that a molecule of heparin links together two heterodimers of FGF and FGF-R into a highly asymmetrical pentameric complex [18]

Methods

Results

Conclusion