Abstract
Here we introduce the fully quantified spectral imaging (FSI) method as a new tool to probe the stoichiometry and stability of protein complexes in biological membranes. The FSI method yields two dimensional membrane concentrations and FRET efficiencies in native plasma membranes. It can be used to characterize the association of membrane proteins: to differentiate between monomers, dimers, or oligomers, to produce binding (association) curves, and to measure the free energies of association in the membrane. We use the FSI method to study the lateral interactions of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), a member of the receptor tyrosine kinase (RTK) superfamily, in plasma membranes, in vivo. The knowledge gained through the use of the new method challenges the current understanding of VEGFR2 signaling.
Highlights
The function of membrane proteins is often regulated through protein–protein interactions in cellular membranes.[1]
We use the fully quantified spectral imaging (FSI) method to study the lateral interactions between VEGFR2 EC + TM constructs in which the intracellular domain has been substituted with either mTurquoise or yellow fluorescent protein (YFP), a FRET pair of fluorescent proteins.[40,41]
The crystal structure of the isolated D7 VEGFR2 EC domains (PDB entry 3KQV) shows a very prominent salt bridge between residues D731 and R726, joining two adjacent molecules in the dimer.[19]. To study if this salt bridge plays a role in VEGFR2 EC + TM dimer stabilization in the absence of ligand, we introduced a D731A mutation in VEGFR2 EC + TM and measured the self-association of this mutant in the plasma membrane with the FSI method. 278 swollen cells expressing the D731A mutant were imaged, and 523 B 3 mm membrane regions in these cells were analyzed to yield FRET efficiencies, donor concentrations, and acceptor concentrations
Summary
The function of membrane proteins is often regulated through protein–protein interactions in cellular membranes.[1]. Endothelial Growth Factor Receptor 2 (VEGFR2), a member of the receptor tyrosine kinase (RTK) superfamily, in plasma membranes of live cells. RTKs epitomize functional regulation through lateral interaction in the plasma membrane.[7,8] RTK dimerization is necessary for RTK function, as contacts between the intracellular kinase domains lead to kinase cross-phosphorylation and initiation of downstream signaling cascades that control cell growth, differentiation and motility during development and in the adult, and in many pathologies such as cancers and growth disorders.[7] While RTK dimerization has been long assumed to occur in response to ligand binding,[9] recent work has suggested that RTKs have a propensity to interact even in the absence of bound ligand, challenging the canonical view of RTK ligand-induced dimerization and activation.[10,11,12] Yet, the mode of activation of many RTKs has far remained controversial, and is still a topic of intense investigations and debate
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