RESUMO
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.
Assuntos
Multimerização Proteica , Receptor EphA2 , Receptor EphA2/metabolismo , Receptor EphA2/química , Receptor EphA2/genética , Humanos , Transferência Ressonante de Energia de Fluorescência , Membrana Celular/metabolismo , Conformação Proteica em alfa-Hélice , MutaçãoRESUMO
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.
RESUMO
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.
Assuntos
Proteínas de Membrana , Microscopia de Fluorescência , Proteínas de Membrana/metabolismo , Proteínas de Membrana/química , Microscopia de Fluorescência/métodos , Humanos , Membrana Celular/metabolismo , Membrana Celular/química , Ligação Proteica , AnimaisRESUMO
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.
Assuntos
Fatores de Crescimento de Fibroblastos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos , Transdução de Sinais , Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Humanos , Ligantes , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Condrócitos/metabolismoRESUMO
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.
Assuntos
Meliteno , Simulação de Dinâmica Molecular , Nanoporos , Fosfatidilcolinas , Meliteno/química , Fosfatidilcolinas/química , Bicamadas Lipídicas/química , Ligação de Hidrogênio , Peptídeos/química , HumanosRESUMO
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.