Strong dimerization of wild-type ErbB2/Neu transmembrane domain and the oncogenic Val664Glu mutant in mammalian plasma membranes

Placone, Jesse K.; He, Lijuan; Piccolo, Nuala Del; Hristova, Kalina

doi:10.1016/j.bbamem.2014.03.001

Cited by 15 publications

(14 citation statements)

References 54 publications

(67 reference statements)

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“…They were imaged using a Nikon Eclipse C1 laser scanning confocal microscope. Each vesicle was imaged using three separate scans: a donor scan, a FRET scan, and an acceptor scan, as described in previous publications (25,(33)(34)(35)(36). Following image acquisition, vesicles were processed using an in-house MATLAB script (21).…”

Section: Resultsmentioning

confidence: 99%

Effect of Thanatophoric Dysplasia Type I Mutations on FGFR3 Dimerization

Piccolo

Placone

Hristova

2015

Biophysical Journal

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Thanatophoric dysplasia type I (TDI) is a lethal human skeletal growth disorder with a prevalence of 1 in 20,000 to 1 in 50,000 births. TDI is known to arise because of five different mutations, all involving the substitution of an amino acid with a cysteine in fibroblast growth factor receptor 3 (FGFR3). Cysteine mutations in receptor tyrosine kinases (RTKs) have been previously proposed to induce constitutive dimerization in the absence of ligand, leading to receptor overactivation. However, their effect on RTK dimer stability has never been measured experimentally. In this study, we characterize the effect of three TDI mutations, Arg248Cys, Ser249Cys, and Tyr373Cys, on FGFR3 dimerization in mammalian membranes, in the absence of ligand. We demonstrate that the mutations lead to surprisingly modest dimer stabilization and to structural perturbations of the dimers, challenging the current understanding of the molecular interactions that underlie TDI.

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Section: Resultsmentioning

confidence: 99%

Effect of Thanatophoric Dysplasia Type I Mutations on FGFR3 Dimerization

Piccolo

Placone

Hristova

2015

Biophysical Journal

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“…Recent studies showed that a somatic mutation destabilizes dimer formation [49], while in another case, a disease-causing mutation was demonstrated to stabilize the dimer [50]. Missense mutations associated with cancer [51], Snyder-Robinson syndrome [19], and congenital heart diseases [52] were linked with altered protein-protein and protein-DNA interactions. In general, any deviation in affinity caused by mutation(s), enhanced or reduced, possesses a high risk of developing a disease.…”

Section: Impact Of Mutations On Protein-protein Protein-ligand and Pmentioning

confidence: 99%

Structural and physico-chemical effects of disease and non-disease nsSNPs on proteins

Kucukkal

Petukh

et al. 2015

Current Opinion in Structural Biology

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This review emphasizes the effects of naturally occurring mutations on structural features and physico-chemical properties of proteins. The basic protein characteristics considered are stability, dynamics, and the binding of proteins and methods for assessing effects of mutations on these macromolecular characteristics are briefly outlined. It is emphasized that the above entities mostly reflect global characteristics of considered macromolecules, while given mutations may alter the local structural features such as salt bridges and hydrogen bonds without affecting the global ones. Furthermore, it is pointed out that disease-causing mutations frequently involve a drastic change of amino acid physico-chemical properties such as charge, hydrophobicity, and geometry, and are less surface exposed than polymorphic mutations.

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“…If the proteins exist in a monomer-dimer equilibrium, the broad range of concentrations is required such that the association model can be fitted to the data. In the case of constitutively dimeric receptors, data over a broad range is required so we can convince ourselves that the receptors are indeed 100% dimeric, by the lack of dependence of FRET on concentration 7 . In this case, the measured FRET depends only on the Intrinsic FRET and the acceptor fraction.…”

Section: Technical Advantages Of the Qi-fret Methods In Rtk Researchmentioning

confidence: 99%

“…In this case, the measured FRET depends only on the Intrinsic FRET and the acceptor fraction. Since the acceptor fraction is known, the Intrinsic FRET can be directly determined for constitutive dimers 7 . In this case, the QI-FRET method can be used as a structural assay.…”

Section: Technical Advantages Of the Qi-fret Methods In Rtk Researchmentioning

confidence: 99%

“…The QI-FRET method yields binding curves and equilibrium constants in native membrane environments, for membrane proteins that have been labeled with donor and acceptor fluorescent proteins (a FRET pair) 4,5,6,7,8 . Here we also overview recently acquired basic knowledge about the second largest class of membrane receptors, the receptor tyrosine kinases (RTKs), which has been gained through the use of the QI-FRET methodology.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of Membrane Protein Interactions in Plasma Membrane Derived Vesicles with Quantitative Imaging Förster Resonance Energy Transfer

2015

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CONSPECTUS Here we describe an experimental tool, termed Quantitative Imaging Förster Resonance Energy Transfer (QI-FRET), which enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles which bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), an RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor many pathogenic single amino acid mutations that cause skeletal and cranial dysplasias, as well as cancer, we also study the effects of these mutations on dimerization. First, we show that the A391E mutation, linked to Crouzon syndrome with acanthosis nigricans, and to bladder cancer, significantly enhances FGFR3 dimerization in the absence of ligand and thus induces aberrant receptor interactions. Second, we present results about the effect of three cysteine mutations that cause thanatophoric dysplasia, a lethal phenotype. Such cysteine mutations have been hypothesized previously to cause constitutive dimerization, but we find instead that they have a surprisingly modest effect on dimerization. Most of the studied pathogenic mutations also altered FGFR3 dimer structure, suggesting that both increases in dimerization propensities and changes in dimer structure contribute to the pathological phenotypes. The results acquired with the QI-FRET method further our understanding of the interactions between FGFR3 molecules, and RTK molecules in general. Since RTK ...

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Strong dimerization of wild-type ErbB2/Neu transmembrane domain and the oncogenic Val664Glu mutant in mammalian plasma membranes

Cited by 15 publications

References 54 publications

Effect of Thanatophoric Dysplasia Type I Mutations on FGFR3 Dimerization

Effect of Thanatophoric Dysplasia Type I Mutations on FGFR3 Dimerization

Structural and physico-chemical effects of disease and non-disease nsSNPs on proteins

Characterization of Membrane Protein Interactions in Plasma Membrane Derived Vesicles with Quantitative Imaging Förster Resonance Energy Transfer

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