Fully quantified spectral imaging reveals<i>in vivo</i>membrane protein interactions

King, Christopher R.; Stoneman, Michael R.; Raicu, Valerică; Hristova, Kalina

doi:10.1039/c5ib00202h

Cited by 88 publications

(296 citation statements)

References 56 publications

(127 reference statements)

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“…Biophysical information about drug targets can yield better mechanistic understanding of their function and ultimately can allow for more accurate prediction of drug action and function. Recently, advances in microscopy have complemented the biochemical assays traditionally used in membrane protein research, producing new insights about membrane protein function (6,7,11,23,24,43,(55)(56)(57). In this paper, we build on our previous work to develop and implement a quantitative FRET assay for examining RTK heterodimers.…”

Section: Discussionmentioning

confidence: 99%

A New Method to Study Heterodimerization of Membrane Proteins and Its Application to Fibroblast Growth Factor Receptors

Piccolo

Sarabipour

Hristova

2017

Journal of Biological Chemistry

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Edited by Paul E. FraserThe activity of receptor tyrosine kinases (RTKs) is controlled through their lateral association in the plasma membrane. RTKs are believed to form both homodimers and heterodimers, and the different dimers are believed to play unique roles in cell signaling. However, RTK heterodimers remain poorly characterized, as compared with homodimers, because of limitations in current experimental methods. Here, we develop a FRETbased methodology to assess the thermodynamics of hetero-interactions in the plasma membrane. To demonstrate the utility of the methodology, we use it to study the hetero-interactions between three fibroblast growth factor receptors-FGFR1, FGFR2, and FGFR3-in the absence of ligand. Our results show that all possible FGFR heterodimers form, suggesting that the biological roles of FGFR heterodimers may be as significant as the homodimer roles. We further investigate the effect of two pathogenic point mutations in FGFR3 (A391E and G380R) on heterodimerization. We show that each of these mutations stabilize most of the heterodimers, with the largest effects observed for FGFR3 wild-type/mutant heterodimers. We thus demonstrate that the methodology presented here can yield new knowledge about RTK interactions and can further our understanding of signal transduction across the plasma membrane. Receptor tyrosine kinases (RTKs)2 regulate many key biological processes, including cell survival, growth, differentiation, and migration. There are 58 different RTKs, classified into 20 families based on sequence similarity. An archetypal RTK consists of a ligand-binding extracellular domain, a single-pass transmembrane (TM) domain, and an intracellular (IC) kinase domain (1-5). These receptors are activated upon dimerization, which is known to be a reversible process (6, 7). Dimer formation is required (although not sufficient) for function (2, 6, 8 -11), because it brings the two kinases into close proximity, enabling cross-phosphorylation on specific tyrosines. Phosphorylated RTKs trigger many intracellular signaling cascades, including the MAPK, PI3K, PKC, and STAT pathways. These pathways, in turn, determine cell fate and function (1-5, 12, 13).RTKs play a fundamental role in human development. They are also critical players in the induction and progression of many cancers (1-5, 13-15). Thus, significant efforts have been dedicated to the development of RTK-specific therapies with high specificity and low toxicity. One class of anti-cancer drugs on the market specifically aims to inhibit RTK dimerization, because it is an important regulator of function. The best known example of these drugs is Herceptin, an antibody raised against the extracellular domain of HER2, which is often overexpressed in breast cancer (15, 16). Although Herceptin treatment can significantly improve patient outcomes in some cases, the performance of this treatment and other RTK-targeted molecular therapies has not reached expectations (4,16,17). This may be partly due to gaps in basic knowledge about RTK intera...

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Section: Discussionmentioning

confidence: 99%

A New Method to Study Heterodimerization of Membrane Proteins and Its Application to Fibroblast Growth Factor Receptors

Piccolo

Sarabipour

Hristova

2017

Journal of Biological Chemistry

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“…Protoplasts transfected by single plasmids were used as controls. Acquisition of spectrally resolved fluorescence images, spectral unmixing of donor, acceptor, and autofluorescence signals, as well as determination of FRET efficiency were performed as described previously (Raicu and Singh, 2013;King et al, 2016). Briefly, cells coexpressing EMS1-CFP and SERK1-YFP (controls: cells expressing only EMS1-CFP or SERK1-YFP) were imaged using an optical microspectroscope (OptiMiS) with the line-scan excitation powered by a femtosecond laser emitting near-infrared light pulses with tunable emission wavelength between 780 and 1,040 nm (Raicu et al, 2009;Biener et al, 2013).…”

Section: Fret Assaymentioning

confidence: 99%

Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination

Wang

Huang

et al. 2016

Plant Physiol.

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Cell signaling pathways mediated by leucine-rich repeat receptor-like kinases (LRR-RLKs) are essential for plant growth, development, and defense. The EMS1 (EXCESS MICROSPOROCYTES1) LRR-RLK and its small protein ligand TPD1 (TAPETUM DETERMINANT1) play a fundamental role in somatic and reproductive cell differentiation during early anther development in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether other cell surface molecules serve as coregulators of EMS1. Here, we show that SERK1 (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1) and SERK2 LRR-RLKs act redundantly as coregulatory and physical partners of EMS1. The SERK1/2 genes function in the same genetic pathway as EMS1 in anther development. Bimolecular fluorescence complementation, Förster resonance energy transfer, and coimmunoprecipitation approaches revealed that SERK1 interacted biochemically with EMS1. Transphosphorylation of EMS1 by SERK1 enhances EMS1 kinase activity. Among 12 in vitro autophosphorylation and transphosphorylation sites identified by tandem mass spectrometry, seven of them were found to be critical for EMS1 autophosphorylation activity. Furthermore, complementation test results suggest that phosphorylation of EMS1 is required for its function in anther development. Collectively, these data provide genetic and biochemical evidence of the interaction and phosphorylation between SERK1/2 and EMS1 in anther development.

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“…Such specific interactions between the two D7 domains could stabilize VEGFR2 unliganded dimers. In our previous work, we therefore asked if the D731-R726 salt bridge plays a role in dimer stabilization, in the absence of ligand (18). We reasoned that if this salt bridge forms in the absence of ligand and if it stabilizes the dimer, its elimination will reduce, or even eliminate, VEGFR2 dimerization.…”

Section: Introductionmentioning

confidence: 99%

“…If, however, the salt bridge does not form and plays no role in unliganded dimerization, its elimination will have no effect on VEGFR2 dimerization. To investigate which of these possibilities occur, we introduced a D731A mutation in a VEGFR2 construct in which the intracellular domain was substituted with a fluorescent protein to allow for FRET detection, and we assessed the effect of the D731A mutation on its dimerization in the absence of ligand (18). To our surprise, none of the two hypothesized scenarios occurred in these experiments.…”

Section: Introductionmentioning

confidence: 99%

“…The approach that we take is to utilize serial deletions of specific Ig-like domains of VEGFR2 EC-TM constructs. The interactions between several truncated versions of the VEGFR2 receptor are studied using two photon microscopy and the recently introduced Fully Quantified Spectral Imaging (FSI) method (18). This method yields approximation-free measurements of the apparent FRET efficiency as well as receptor concentrations in the plasma membrane.…”

Section: Introductionmentioning

confidence: 99%

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Cooperative interactions between VEGFR2 extracellular Ig-like subdomains ensure VEGFR2 dimerization

King

Wirth

Workman

et al. 2017

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background Prior studies have suggested that the interactions occurring between VEGFR2 extracellular domains in the absence of ligand are complex. Here we seek novel insights into these interactions, and into the role of the different Ig-like domains (D1 through D7) in VEGFR2 dimerization. Methods We study the dimerization of a single amino acid mutant and of three deletion mutants in the plasma membrane using two photon microscopy and fully quantified spectral imaging. Results We demonstrate that a set of cooperative interactions between the different Ig-like domains ensure that VEGFR2 dimerizes with a specific affinity instead of forming oligomers. Conclusions The contributions of subunits D7 and D4 seem to be the most critical, as they appear essential for strong lateral interactions and for the formation of dimers, respectively. General Significance This study provides new insights into the mechanism of VEGFR2 dimerization and activation.

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Fully quantified spectral imaging revealsin vivomembrane protein interactions

Cited by 88 publications

References 56 publications

A New Method to Study Heterodimerization of Membrane Proteins and Its Application to Fibroblast Growth Factor Receptors

A New Method to Study Heterodimerization of Membrane Proteins and Its Application to Fibroblast Growth Factor Receptors

Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination

Cooperative interactions between VEGFR2 extracellular Ig-like subdomains ensure VEGFR2 dimerization

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