Transmembrane Helix Heterodimerization in Lipid Bilayers: Probing the Energetics behind Autosomal Dominant Growth Disorders

Merzlyakov, Mikhail; You, Min; Li, Edwin; Hristova, Kalina

doi:10.1016/j.jmb.2006.01.086

Cited by 34 publications

(35 citation statements)

References 27 publications

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“…The corrected FRET efficiency, E D , is due to heterodimerization only. FRET efficiency is measured through the change in donor fluorescence, so E D can be written in terms of the concentration of the acceptor-donor complex (that is, the heterodimer) and the concentration of the donor (38,44,45),…”

Section: Resultsmentioning

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: Resultsmentioning

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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“…For intrapentameric FRET experiments, each cell's relative protein expression was assessed as a sum of the starting YFP fluorescence (prebleach) and the final CFP fluorescence after FRET was abolished (postbleach). In general, concentrations of membrane proteins and related dissociation constants (K D ) are expressed as protein molar fraction (20) or as species per unit area (e.g. mol/m 2 ).…”

Section: Methodsmentioning

confidence: 99%

Phospholamban Oligomerization, Quaternary Structure, and Sarco(endo)plasmic Reticulum Calcium ATPase Binding Measured by Fluorescence Resonance Energy Transfer in Living Cells

Kelly

Hou

Bossuyt

et al. 2008

Journal of Biological Chemistry

116

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Phospholamban (PLB) oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase (SERCA) binding were quantified by fluorescence resonance energy transfer (FRET) in an intact cellular environment. FRET between cyan fluorescent protein-PLB and yellow fluorescent protein-PLB in AAV-293 cells showed hyperbolic dependence on protein concentration, with a maximum efficiency of 45.1 ؎ 1.3%. The observed FRET corresponds to a probe separation distance of 58.7 ؎ 0.5 Å , according to a computational model of intrapentameric FRET. This is consistent with models of the PLB pentamer in which cytoplasmic domains fan out from the central bundle of transmembrane helices. An I40A mutation of PLB did not alter pentamer conformation but increased the concentration of half-maximal FRET (K D ) by >4-fold. This is consistent with the previous observation that this putatively monomeric mutant still oligomerizes in intact membranes but forms more dynamic pentamers than wild type PLB. PLB association with SERCA, measured by FRET between cyan fluorescent protein-SERCA and yellow fluorescent protein-PLB, was increased by the I40A mutation without any detectable change in probe separation distance. The data indicate that the regulatory complex conformation is not altered by the I40A mutation. A naturally occurring human mutation (L39Stop) greatly reduced PLB oligomerization and SERCA binding and caused mislocalization of PLB to the cytoplasm and nucleus. Overall, the data suggest that the PLB pentamer adopts a "pinwheel" shape in cell membranes, as opposed to a more compact "bellflower" conformation. I40A mutation decreases oligomerization and increases PLB binding to SERCA. Truncation of the transmembrane domain by L39Stop mutation prevents anchoring of the protein in the membrane, greatly reducing PLB binding to itself or its regulatory target, SERCA.The 52-amino acid protein phospholamban (PLB) 2 is an important regulator of cardiac calcium handling (1). PLB binds avidly (2) but reversibly (3) to the sarco(endo)plasmic reticulum calcium ATPase (SERCA), reducing this calcium pump's affinity for calcium (4). PLB also oligomerizes into pentamers through leucine zipper interactions in its transmembrane domain (5, 6). NMR studies indicate that PLB tertiary structure consists of an N-terminal (cytosolic) ␣-helix (domain IA) connected by a flexible loop (domain IB) to a second ␣-helix (domain II) that is anchored in the membrane of the sarcoplasmic reticulum (7). Several possible configurations of the cytoplasmic domains of pentameric PLB have been described. Some of these portray the domain IA ␣-helix as being nearly normal to the surface of the membrane (8, 9), whereas others indicate axial declination of domain IA (7, 10 -14), permitting contact with the surface of the membrane (15, 16). These previous studies were performed using in vitro preparations of PLB in defined lipids or detergent. To investigate the quaternary structure of PLB in the membranes of living cells and distinguish between these structural mo...

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“…A method to measure thermodynamics of homo and heterodimerization of TM helices in lipid bilayers is FRET. 25,26 Experimental details associated with such measurements were recently reviewed. 27 The advantage of this method over the bacterial assays is that it can directly measure dimer fractions and yield dimerization free energies.…”

Section: Thermodynamics Of Rtk Tm Domain Dimerizationmentioning

confidence: 99%

Receptor tyrosine kinase transmembrane domains

Hristova

2010

Cell Adhesion & Migration

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Transmembrane Helix Heterodimerization in Lipid Bilayers: Probing the Energetics behind Autosomal Dominant Growth Disorders

Cited by 34 publications

References 27 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

Phospholamban Oligomerization, Quaternary Structure, and Sarco(endo)plasmic Reticulum Calcium ATPase Binding Measured by Fluorescence Resonance Energy Transfer in Living Cells

Receptor tyrosine kinase transmembrane domains

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