Signaling networks: Talking in detail

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Analysis of the regulatory cross-talk occurring between 22 receptor-specific ligands provides new insights into context-dependent signaling.

Although cells transduce environmental signals through a limited set of canonical signaling pathways, the pathways themselves are activated by a sizeable array of ligands. Combinations of these ligands can generate a vast number of inputs; for example, 20 different ligands might in principle elicit over a million possible unique output responses. The Alliance for Cellular Signaling (AfCS) set out to examine the complexity of these responses by analyzing the level of cross-talk between 22 receptor-specific ligands in RAW 264.7 macrophages. This study, published in Nature Cell Biology, reveals novel signaling circuits and provides insight into context-dependent signaling by providing an estimate of the level of regulatory cross-talk in this system.

Ranganathan and colleagues selected representative ligands of diverse signaling pathways and measured signaling parameters such as cAMP synthesis, protein phosphorylation and intracellular calcium mobilization, as well as signaling outputs such as cytokine secretion. To test the robustness of their approach the authors constructed a clustered matrix representing the input and output responses for all 22 single ligands. This single ligand matrix was largely consistent with published signaling mechanisms.

A similar matrix of all 231 pair-wise ligand combinations was then assembled. Where no cross-talk occurs, the expected output of two ligands is simply additive. An output higher than this would indicate cross-talk and synergy, possibly via direct interaction or feedback mechanisms. This screen yielded predicted cases of synergistic signaling as well as several novel interactions. For example, combinations of Ca²⁺-mobilizing and cAMP-inducing ligands led to synergistic increases in cAMP in conjunction with the synergistic inhibition of Ca²⁺ mobilization. Cells treated with ionomycin, to raise levels of Ca²⁺ or EGTA to chelate intracellular calcium, revealed that Ca²⁺ mobilization alone is insufficient to support synergistic signaling. Ligand-mediated cAMP production is specifically upregulated by receptor-mediated calcium mobilization, representing a novel Ca²⁺-cAMP signaling circuit.

All ligands diplayed non-additive, synergistic activatory or inhibitory responses in different pair-wise combinations, with the majority of the ligand pairs’ output falling into a relatively small number of clusters, possibly representing pathway convergence. For example, the screen showed that Toll like receptor (TLR) signaling is modulated by a cAMP-dependent process. This is in agreement with published information that Gαs signaling can attenuate inflammatory responses due to TLR activity.

This study shows how a relatively small number of ligands are able to regulate downstream interactions in a context-dependent manner. The emerging basic architecture of the signaling network for the RAW 264.7 macrophage cell line can be expected to provide an overview of signaling cross-talk in other mammalian systems.

Clare Garvey
Signaling Gateway

References

Madhusudan Natarajan, Keng-Mean Lin, Robert C. Hsueh, Paul C. Sternweis & Rama Ranganathan. A global analysis of cross-talk in a mammalian cellular signalling network. Nature Cell Biology 8, 571 - 580 (2006); | Article | PubMed |