Flim- FRET, a Structural Tool for ErbB Receptor Studies in the Living Cell

Abstract

The association state(s) and activities of the ErbB receptor family members in intact living cells differ widely depending upon expression levels and their distribution and interaction partners. There are contradictory views in the literature about the aggregation states and presumed structures of the receptors in the cell membrane. Fixation artifacts may account for apparent quantitative discrepancies. We obtained biophysical FRET/FLIM data on living cells that reveal structural features of ErbB1 (EGFR) and ErbB2 as well as the effects of EGF and various kinase inhibitors on these structures.We constructed transgenes in which an acyl carrier protein sequence was introduced between the signal peptide and the mature receptor protein sequence. ACP-ErbB1 behaves similarly to wild type ErbB1 with respect to EGF binding, activation and internalization. The ACP-ErbB2 lacks the capacity for binding ligands but can be transactivated as a heterodimer with ErbB1 or ErbB3. Enzymatic labeling of the specific serine in the ACP tag by fluorescent CoA substrates served as donors. The FRET acceptor was the novel membrane probe, NR12S, which is confined exclusively to the outer leaflet of the plasma membrane.Addition of NR12S to the cells led to a dramatic reduction in the fluorescence lifetime of the donor, indicating a close proximity of the N-terminus of the ErbB1 ectodomain to the plasma membrane, supporting the published autoinhibited structure. EGF addition caused a time-dependent increase in the donor lifetime (reduced FRET), in accordance with the extended dimeric ectodomain structure oberved by Xray-crystallography. The effects of kinase inhibitors on these states and on ensuing endocytosis were also studied. The influence(s) of ErB2 density and antibodies interfering with receptor dimerization were additional topics addressed in this study. TCSPC lifetime images were analyzed with Mathematica software developed for these studies.