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Signaling bias is the ability of a receptor to differentially activate downstream signaling pathways in response to different ligands. Bias investigations have been hindered by inconsistent results in different cellular contexts. Here we introduce a methodology to identify and quantify bias in signal transduction across the plasma membrane without contributions from feedback loops and system bias. We apply the methodology to quantify phosphorylation efficiencies and determine absolute bias coefficients. We show that the signaling of epidermal growth factor receptor (EGFR) to EGF and TGFα is biased towards Y1068 and against Y1173 phosphorylation, but has no bias for epiregulin. We further show that the L834R mutation found in non-small-cell lung cancer induces signaling bias as it switches the preferences to Y1173 phosphorylation. The knowledge gained here challenges the current understanding of EGFR signaling in health and disease and opens avenues for the exploration of biased inhibitors as anti-cancer therapies. 

The differential signaling of multiple FGF ligands through a single fibroblast growth factor (FGF) receptor (FGFR) plays an important role in embryonic development. Here, we use quantitative biophysical tools to uncover the mechanism behind differences in FGFR1c signaling in response to FGF4, FGF8, and FGF9, a process which is relevant for limb bud outgrowth. We find that FGF8 preferentially induces FRS2 phosphorylation and extracellular matrix loss, while FGF4 and FGF9 preferentially induce FGFR1c phosphorylation and cell growth arrest. Thus, we demonstrate that FGF8 is a biased FGFR1c ligand, as compared to FGF4 and FGF9. Förster resonance energy transfer experiments reveal a correlation between biased signaling and the conformation of the FGFR1c transmembrane domain dimer. Our findings expand the mechanistic understanding of FGF signaling during development and bring the poorly understood concept of receptor tyrosine kinase ligand bias into the spotlight. 

Peptides that form nanoscale pores in lipid bilayers have potential applications in triggered release, but only if their selectivity for target synthetic membranes over bystander biomembranes can be optimized. Previously, we identified a novel family of α-helical pore-forming peptides called “macrolittins”, which release macromolecular cargoes from phosphatidylcholine (PC) liposomes at concentrations as low as 1 peptide per 1000 lipids. In this work, we show that macrolittins have no measurable cytolytic activity against multiple human cell types even at high peptide concentration. This unprecedented selectivity for PC liposomes over cell plasma membranes is explained, in part, by the sensitivity of macrolittin activity to physical chemical properties of the bilayer hydrocarbon core. In the presence of cells, macrolittins release all vesicle-entrapped cargoes (proteins and small molecule drugs) which are then readily uptaken by cells. Triggered release occurs without any direct effect of the peptide on the cells, and without vesicle–vesicle or vesicle–cell interactions.