Direct Assessment of the Effect of the Gly380Arg Achondroplasia Mutation on FGFR3 Dimerization Using Quantitative Imaging FRET

Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland, United States of America.
PLoS ONE (Impact Factor: 3.23). 10/2012; 7(10):e46678. DOI: 10.1371/journal.pone.0046678
Source: PubMed


The Gly380Arg mutation in FGFR3 is the genetic cause for achondroplasia (ACH), the most common form of human dwarfism. The mutation has been proposed to increase FGFR3 dimerization, but the dimerization propensities of wild-type and mutant FGFR3 have not been compared. Here we use quantitative imaging FRET to characterize the dimerization of wild-type FGFR3 and the ACH mutant in plasma membrane-derived vesicles from HEK293T cells. We demonstrate a small, but statistically significant increase in FGFR3 dimerization due to the ACH mutation. The data are consistent with the idea that the ACH mutation causes a structural change which affects both the stability and the activity of FGFR3 dimers in the absence of ligand.

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    • "These vesicles are derived from the native cellular membrane, and are thus more native-like than vesicles made of synthetic lipids. They are increasingly used in studies of lipid–lipid, lipid–protein and protein–protein interactions, and have already yielded new knowledge about lipid domains and receptor interactions in the membrane [11] [12] [13] [14] [15]. Often, however, they are not well characterized in terms of their lipid and protein content. "
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    ABSTRACT: Plasma membrane-derived vesicles are being used in biophysical and biochemical research as a simple, yet native-like model of the cellular membranes. Here we report on the characterization of vesicles produced via two different vesiculation methods from CHO and A431 cell lines. The first method is a recently developed method which utilizes chloride salts to induce osmotic vesiculation. The second is a well established chemical vesiculation method which uses DTT and formaldehyde. We show that both vesiculation methods produce vesicles which contain the lipid species previously reported in the plasma membrane of these cell lines. The two methods lead to small but statistically significant differences in two lipid species only; phosphatidylcholine (PC) and plasmalogen phosphatidylethanolamine (PEp). However, highly significant differences were observed in the degree of incorporation of a membrane receptor and in the degree of retention of soluble cytosolic proteins within the vesicles. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Apr 2015 · Biochimica et Biophysica Acta
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    • "The plasma membrane derived vesicles lack a transmembrane potential and a cytoskeleton, and are not a perfect mimic of the plasma membrane. Yet, plasma membrane derived vesicles are the only model system that has allowed us thus far to characterize the interaction strength between glycosylated membrane proteins in quantitative terms [16] [23]. "
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    ABSTRACT: Membrane protein interactions, which underlie biological function, take place in the complex cellular membrane environment. Plasma membrane derived vesicles are a model system which allows the interactions between membrane proteins to be studied without the need for their extraction, purification, and reconstitution into lipid bilayers. Plasma membrane vesicles can be produced from different cell lines and by different methods, providing a rich variety of native-like model systems. With these choices, however, questions arise as to how the different types of vesicle preparations affect the interactions between membrane proteins. Here we address this question using the glycophorin A transmembrane domain (GpA) as a model system. We compare the dimerization of GpA in six different vesicle preparations derived from CHO, HEK 293T and A431 cells. We accomplish this with the use of a FRET-based method which yields the FRET efficiency, the donor concentration, and the acceptor concentration in each vesicle. We show that the vesicle preparation protocol has no statistically significant effect on GpA dimerization. Based on these results, we propose that any of six the plasma membrane preparations investigated here can be used as a model system for studies of membrane protein interactions.
    Full-text · Article · Apr 2013 · Biochimica et Biophysica Acta

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