The Dipole Potential Influences the Clustering of ErbB Proteins

Abstract

ErbB proteins are receptor tyrosine-kinases and their signal transduction mechanisms play significant roles in the pathogenesis of tumors. Their homo- and heteroassociations lead to activation of signaling. The role of the extracellular domain is well-known in regulating receptor clustering but the transmembrane domain may also play a role. Dipole potential is a large positive potential barrier at the membrane midplane created by inward-pointing molecular dipoles at the interfacial planes and it might influence the functions of transmembrane domains of receptors.6-ketocholestanol and phloretin were used to increase and decrease, respectively, the dipole potential. Their effect was evaluated by the dipole potential sensitive fluorescent dye, di-8-ANEPPS. The homo- and heteroassociations of ErbB proteins in serum-starved and EGF-stimulated SKBR-3 cells were measured by flow cytometric fluorescence resonance energy transfer and by fluorescence microscopy using number&brightness analysis. The functional effect of changing dipole potential was determined in both starved and stimulated cells using flow cytometry by indirect labeling of tyrosine-phosphorylated proteins by PY99, phosphorylated ErbB2 by Ab18 and phosphorylated ErbB1 by anti-pEGFR antibodies.The dipole potential was successfully increased by 6-ketocholestanol and decreased by phloretin in SKBR-3, JIMT-1 and CHO cell lines. An increased dipole potential resulted in a significant increase in ErbB2-ErbB2 homoassociation both in starved and EGF stimulated samples, it increased ErbB1-ErbB1 homo- and ErbB1-ErbB2 heteroassociation in stimulated cells, while decreasing the dipole potential caused a non-significant decrease in most samples. The effect on homoassociations was confirmed by number&brightness analysis. Ketocholestanol increased and phloretin decreased ErbB2-, ErbB1-specific and general tyrosine phosphorylation in EGF stimulated cells.The dipole potential may play an important role in controlling the homo- and heteroassociation of transmembrane receptors. Intentional or accidental modification of the dipole potential by drugs might result in modified signal transduction processes.