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

Abstract

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 ...

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

“…If these two receptors form heterodimers, FRET will occur. The FRET efficiency can be measured with the QI-FRET method as described (23,24). The measured FRET efficiency can then be corrected for the so-called proximity FRET, to yield the interaction-specific FRET efficiency, E D .…”

“…Given an intrinsic FRET value, Equation 10 can be solved for d to estimate fluorophore separation in a given dimer. Using QI-FRET, we can measure three values per vesicle: donor concentration, acceptor concentration, and FRET efficiency (23,24). Because one receptor is labeled with the donor, and the other receptor is labeled with the acceptor, the receptor concentrations, [X total ] and [Y total ], can be directly measured as the donor concentration and acceptor concentration.…”

“…Twenty-four hours post-transfection, the cells are exposed to an osmotic stress buffer (22) that produces plasma membrane-derived vesicles. These vesicles are collected and then imaged using the QI-FRET method (23,24). In brief, a confocal microscope is used to image the cross-section of each vesicle in the donor, FRET, and acceptor channels (see Fig.…”

“…This variation explains the seemingly wide spread of FRET efficiencies seen in the FRET efficiency versus acceptor concentration plots. As discussed previously, a wide range of donor to acceptor ratios, as well as a wide range of concentrations, is an advantage in the QI-FRET methodology, because it ensures a robust fit of a dimerization model to FRET data (24). 12) to the experimental FRET measurements for each heterodimer pair.…”

“…As the receptors are expressed in cells prior to vesicle production, they undergo all post-translational modifications. FRET is measured with the quantitative imaging-FRET (QI-FRET) method (23,24), which yields donor and acceptor concentrations, in addition to FRET efficiencies, in each vesicle.…”