Quantitative and qualitative differences in the activation of a fibroblast growth factor receptor by different FGF ligands.

The FGF system is the most complex of all receptor tyrosine kinase signaling networks with 18 FGF ligands and four FGFRs that deliver morphogenic signals to pattern most embryonic structures. Even when a single FGFR is expressed in the tissue, different FGFs can trigger dramatically different biological responses via this receptor. Here we show both quantitative and qualitative differences in the signaling of one of the FGF receptors, FGFR1c, in response to different FGFs. We provide an overview of the recent discovery that FGFs engage in biased signaling via FGFR1c. We discuss the concept of ligand bias, which represents qualitative differences in signaling as it is a measure of differential ligand preferences for different downstream responses. We show how FGF ligand bias manifests in functional data in cultured chondrocyte cells. We argue that FGF-ligand bias contributes substantially to FGF-driven developmental processes, along with known differences in FGF expression levels, FGF-FGFR binding coefficients and differences in FGF stability in vivo.

Structural Determinants of Peptide Nanopore Formation.

We have evolved the nanopore-forming macrolittin peptides from the bee venom peptide melittin using successive generations of synthetic molecular evolution. Despite their sequence similarity to the broadly membrane permeabilizing cytolytic melittin, the macrolittins have potent membrane selectivity. They form nanopores in synthetic bilayers made from 1-palmitoyl, 2-oleoyl-phosphatidylcholine (POPC) at extremely low peptide concentrations and yet have essentially no cytolytic activity against any cell membrane, even at high concentration. Here, we explore the structural determinants of macrolittin nanopore stability in POPC bilayers using atomistic molecular dynamics simulations and experiments on macrolittins and single-site variants. Simulations of macrolittin nanopores in POPC bilayers show that they are stabilized by an extensive, cooperative hydrogen bond network comprised of the many charged and polar side chains interacting with each other via bridges of water molecules and lipid headgroups. Lipid molecules with unusual conformations participate in the H-bond network and are an integral part of the nanopore structure. To explore the role of this H-bond network on membrane selectivity, we swapped three critical polar residues with the nonpolar residues found in melittin. All variants have potency, membrane selectivity, and cytotoxicity that were intermediate between a cytotoxic melittin variant called MelP5 and the macrolittins. Simulations showed that the variants had less organized H-bond networks of waters and lipids with unusual structures. The membrane-spanning, cooperative H-bond network is a critical determinant of macrolittin nanopore stability and membrane selectivity. The results described here will help guide the future design and optimization of peptide nanopore-based applications.

Transmembrane helix interactions regulate oligomerization of the receptor tyrosine kinase EphA2.

The transmembrane helices of receptor tyrosine kinases (RTKs) have been proposed to switch between two different dimeric conformations, one associated with the inactive RTK and the other with the active RTK. Furthermore, recent work has demonstrated that some full-length RTKs are associated into oligomers that are larger than dimers, raising questions about the roles of the TM helices in the assembly and function of these oligomers. Here we probe the roles of the TM helices in the assembly of EphA2 RTK oligomers in the plasma membrane. We employ mutagenesis to evaluate the relevance of a published NMR dimeric structure of the isolated EphA2 TM helix in the context of the full-length EphA2 in the plasma membrane. We use two fluorescence methods, Förster Resonance Energy Transfer and Fluorescence Intensity Fluctuations spectrometry, which yield complementary information about the EphA2 oligomerization process. These studies reveal that the TM helix mutations affect the stability, structure, and size of EphA2 oligomers. However, the effects are multifaceted and point to a more complex role of the TM helix than the one expected from the "TM dimer switch" model.

Probing phosphorylation events in biological membranes: The transducer function.

The extracellular environment is sensed by receptors in the plasma membrane. Some of these receptors initiate cytoplasmic signaling cascades involving phosphorylation: the addition of a phosphate group to a specific amino acid, such as tyrosine, in a protein. Receptor Tyrosine Kinases (RTKs) are one large class of membrane receptors that can directly initiate signaling cascades through their intracellular kinase domains, which both catalyze tyrosine phosphorylation and get phosphorylated. In the first step of signaling, the ligands stabilize phosphorylation-competent RTK dimers and oligomers, which leads to the phosphorylation of specific tyrosine residues in the activation loop of the kinases. Here we discuss quantitative measurements of tyrosine phosphorylation efficiencies for RTKs, described by the "transducer function". The transducer function links the phosphorylation (the response) and the binding of the activating ligand to the receptor (the stimulus). We overview a methodology that allows such measurements in direct response to ligand binding. We discuss experiments which demonstrate that EGF is a partial agonist, and that two tyrosines in the intracellular domain of EGFR, Y1068 and Y1173, are differentially phosphorylated in the EGF-bound EGFR dimers.

Ligand bias underlies differential signaling of multiple FGFs via FGFR1.

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.

Investigations of membrane protein interactions in cells using fluorescence microscopy.

The interactions between proteins in membranes govern many cellular functions. Our ability to probe for such interactions has greatly evolved in recent years due to the introduction of new fluorescence techniques. As a result, we currently have a choice of methods that can be used to assess the spatial distribution of a membrane protein, its association state, and the thermodynamic stability of the oligomers in the native milieu. These biophysical measurements have revealed new insights into important biological processes in cellular membranes.

Masking the transmembrane region of the amyloid ß precursor protein as a safe means to lower amyloid ß production.

Introduction: Reducing brain levels of both soluble and insoluble forms of amyloid beta (Aß) remains the primary goal of most therapies that target Alzheimer's disease (AD). However, no treatment has so far resulted in patient benefit, and clinical trials of the most promising drug candidates have generally failed due to significant adverse effects. This highlights the need for safer and more selective ways to target and modulate Aß biogenesis. Methods: Peptide technology has advanced to allow reliable synthesis, purification, and delivery of once-challenging hydrophobic sequences. This is opening up new routes to target membrane processes associated with disease. Here we deploy a combination of atomic detail molecular dynamics (MD) simulations, living-cell Förster resonance energy transfer (FRET), and in vitro assays to elucidate the atomic-detail dynamics, molecular mechanisms, and cellular activity and selectivity of a membrane-active peptide that targets the Aß precursor protein (APP). Results: We demonstrate that Aß biogenesis can be downregulated selectively using an APP occlusion peptide (APPOP). APPOP inhibits Aß production in a dose-dependent manner, with a mean inhibitory concentration (IC50) of 450 nM toward exogenous APP and 50 nM toward endogenous APP in primary rat cortical neuronal cultures. APPOP does not impact the γ-secretase cleavage of Notch-1, or exhibit toxicity toward cultured primary rat neurons, suggesting that it selectively shields APP from proteolysis. Discussion: Drugs targeting AD need to be given early and for very long periods to prevent the onset of clinical symptoms. This necessitates being able to target Aß production precisely and without affecting the activity of key cellular enzymes such as γ-secretase for other substrates. Peptides offer a powerful way for targeting key pathways precisely, thereby reducing the risk of adverse effects. Here we show that protecting APP from proteolytic processing offers a promising route to safely and specifically lower Aß burden. In particular, we show that the amyloid pathway can be targeted directly and specificically. This reduces the risk of off-target effects and paves the way for a safe prophylactic treatment.

Quantification of ligand and mutation-induced bias in EGFR phosphorylation in direct response to ligand binding.

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.

Determining the statistical significance of the difference between arbitrary curves: A spreadsheet method.

We present a simple, spreadsheet-based method to determine the statistical significance of the difference between any two arbitrary curves. This modified Chi-squared method addresses two scenarios: A single measurement at each point with known standard deviation, or multiple measurements at each point averaged to produce a mean and standard error. The method includes an essential correction for the deviation from normality in measurements with small sample size, which are typical in biomedical sciences. Statistical significance is determined without regard to the functionality of the curves, or the signs of the differences. Numerical simulations are used to validate the procedure. Example experimental data are used to demonstrate its application. An Excel spreadsheet is provided for performing the calculations for either scenario.

Utility of FRET in studies of membrane protein oligomerization: The concept of the effective dissociation constant.

The activity of many membrane receptors is controlled through their lateral association into dimers or higher-order oligomers. Although Förster resonance energy transfer (FRET) measurements have been used extensively to characterize the stability of receptor dimers, the utility of FRET in studies of larger oligomers has been limited. Here we introduce an effective equilibrium dissociation constant that can be extracted from FRET measurements for EphA2, a receptor tyrosine kinase (RTK) known to form active oligomers of heterogeneous distributions in response to its ligand ephrinA1-Fc. The newly introduced effective equilibrium dissociation constant has a well-defined physical meaning and biological significance. It denotes the receptor concentration for which half of the receptors are monomeric and inactive, and the other half are associated into oligomers and are active, irrespective of the exact oligomer size. This work introduces a new dimension to the utility of FRET in studies of membrane receptor association and signaling in the plasma membrane.