The epidermal growth factor receptor (EGFR) is activated by dimerization, but activation also generates higher-order multimers, whose nature and function are poorly understood. We have characterized ligand-induced dimerization and multimerization of EGFR using single-molecule analysis, and show that multimerization can be blocked by mutations in a specific region of Domain IV of the extracellular module. These mutations reduce autophosphorylation of the C-terminal tail of EGFR and attenuate phosphorylation of phosphatidyl inositol 3-kinase, which is recruited by EGFR. The catalytic activity of EGFR is switched on through allosteric activation of one kinase domain by another, and we show that if this is restricted to dimers, then sites in the tail that are proximal to the kinase domain are phosphorylated in only one subunit. We propose a structural model for EGFR multimerization through self-association of ligand-bound dimers, in which the majority of kinase domains are activated cooperatively, thereby boosting tail phosphorylation.DOI: http://dx.doi.org/10.7554/eLife.14107.001

No abstract

Plexins are single-pass transmembrane receptors that bind the axon guidance molecules semaphorins. Single-pass transmembrane proteins are an important class of receptors that display a wide variety of activation mechanisms, often involving ligand-dependent dimerization or conformational changes. Resolving the activation mechanism and dimerization state of these receptors is extremely challenging, especially in a live-cell environment. Here, we report on the dimerization state of PlexinA4 and its response to activation by semaphorin binding. Semaphorins are dimeric molecules that activate plexin by binding two copies of plexin simultaneously and inducing formation of a specific active dimer of plexin. An open question is whether there are preexisting plexin dimers that could act as autoinhibitory complexes. We address these questions with pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is a two-color fluorescence microscopy method that is directly sensitive to protein dimerization in a live-cell environment. With PIE-FCCS, we show that inactive PlexinA4 is dimerized in the live-cell plasma membrane. By comparing the cross correlation of full-length PlexinA4 to control proteins and plexin mutants, we show that dimerization of inactive PlexinA4 requires the Sema domain, but not the cytoplasmic domain. Ligand stimulation with Sema6A does not change the degree of cross correlation, indicating that plexin activation does not lead to higher-order oligomerization. Together, the results suggest that semaphorin activates plexin by disrupting an inhibitory plexin dimer and inducing the active dimer.

Fluorescence cross-correlation spectroscopy (FCCS) is an advanced fluorescence technique that can quantify protein-protein interactions in vivo. Due to the dynamic, heterogeneous nature of the membrane, special considerations must be made to interpret FCCS data accurately. In this study, we describe a method to quantify the oligomerization of membrane proteins tagged with two commonly used fluorescent probes, mCherry (mCH) and enhanced green (eGFP) fluorescent proteins. A mathematical model is described that relates the relative cross-correlation value (f) to the degree of oligomerization. This treatment accounts for mismatch in the confocal volumes, combinatoric effects of using two fluorescent probes, and the presence of non-fluorescent probes. Using this model, we calculate a ladder of f values which can be used to determine the oligomer state of membrane proteins from live-cell experimental data. Additionally, a probabilistic mathematical simulation is described to resolve the affinity of different dimeric and oligomeric protein controls.

Binding of biomacromolecules to anionic lipids in the plasma membrane is a common motif in many cell signaling pathways. Previous work has shown that macromolecules with cationic sequences can form nanodomains with sequestered anionic lipids, which alters the lateral distribution and mobility of the membrane lipids. Such sequestration is believed to result from the formation of a lipid-macromolecule complex. To date, however, the molecular structure and dynamics of the lipid-polymer interface are poorly understood. We have investigated the behavior of polycationic quaternized polyvinylpyridine (QPVP) on supported lipid bilayers doped with phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP) lipids using time-resolved fluorescence microscopy, including pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is a dual-color fluorescence spectroscopy that translates fluctuations in fluorescence signal into a measurement of diffusion and colocalization. By labeling the polymer and lipids, we investigated the adsorption-induced translational mobility of lipids and systematically studied the influence of lipid charge density and solution ionic strength. Our results show that alteration of anionic lipid lateral mobility is dependent on the net charge of the lipid headgroup and is modulated by the ionic strength of the solution, indicating that electrostatic interactions drive the decrease in lateral mobility of anionic lipids by adsorbed QPVP. At physiological salt concentration we observe that the lipid lateral mobility is weakly influenced by QPVP and that there is no evidence of stable lipid-polymer complexes.