Investigation of the Dimerization of Proteins from the Epidermal Growth Factor Receptor Family by Single Wavelength Fluorescence Cross-Correlation Spectroscopy

Abstract

Single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS), introduced to study biomolecular interactions, has recently been reported to monitor enzyme activity by using a newly developed fluorescent protein variant together with cyan fluorescent protein. Here, for the first time to our knowledge, SW-FCCS is applied to detect interactions between membrane receptors in vivo by using the widely used enhanced green fluorescent protein and monomeric red fluorescent protein. The biological system studied here is the epidermal growth factor/ErbB receptor family, which plays pivotal roles in the development of organisms ranging from worms to humans. It is widely thought that a ligand binds to the monomeric form of the receptor and induces its dimeric form for activation. By using SW-FCCS and Förster resonance energy transfer, we show that the epidermal growth factor receptor and ErbB2 have preformed homo- and heterodimeric structures on the cell surface and quantitation of dimer fractions is performed by SW-FCCS. These receptors are major targets of anti-cancer drug development, and the receptors' homo- and heterodimeric structures are relevant for such developments.

The Epidermal Growth Factor Receptor Forms Location-Dependent Complexes in Resting Cells

“…The normalized dimer fraction of EGFR was 57% determined by SW-FCCS and DC-FCCS (both at the apical surfaces), and 65% by quasi PIE-FCCS (apical) and imaging FCCS (basal). The normalized values are an estimate of the actual dimer fraction obtained and are comparable with our previous study on EGFR (19). On the other hand, we have observed significant cell-to-cell variability, which was especially obvious from the imaging FCCS experiments.…”

“…Nagy et al (18) observed preformed dimers only at high expression levels of EGFR of ~600,000 receptors per cell. In contrast to that, in our previous study, we found dimerization to be independent of receptor expression levels in the range from 20,000 to 260,000 copies per cell (19). According to single particle tracking studies of EGFR, the receptor forms transient dimers in the absence of EGF (20)(21)(22); on the other hand, results of Tao and Maruyama (23) indicate that EGFR dimers are already formed in the endoplasmic reticulum, which would suggest a much more stable association.…”

“…The background was measured in cells not expressing any fluorescent proteins and illuminated by excitation light of the same intensity as used in the actual SW-FCCS recordings (19). The typical background levels were between 500 and 800 cps.…”

“…The apparent complex fraction of mRFP-EGFR-eGFP in COS-7 and HEK293 cells was 68 5 2% and 58 5 1%, respectively. In our earlier work, we found that the proteins PMT-eGFP/-mRFP do not interact with each other in the membrane in CHO-K1 cells (19), making them a suitable negative control for FCCS measurements in cell membranes. Surprisingly, different behavior of these proteins was observed in COS-7 and HEK293.…”

“…In this work we probe EGFR dimerization by single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS) (19,34) with the aim to understand the potential role of selected experimental factors in the discrepancies in reported levels of EGFR dimerization in resting cells. In particular, we performed all measurements at room as well as at physiological temperature and on the apical as well as on the basal plasma membrane.…”