6th ESACP Congress, Heidelberg, April 7-11, 1999

A157
SMALL AND LARGE SCALE CLUSTERING OF THE ERBB2 RECEPTOR TYROSINE KINASE ON QUIESCENT AND STIMULATED CELLS DETECTED BY SCANNING NEAR-FIELD OPTICAL MICROSCOPY
Nagy P, Jenei A, Kirsch AK, Szollosi J, Damjanovich S

Dept.Biophysics and Cell Biology, University Medical School Debrecen, Hungary, Max Planck Institut für Biophysikalische Chemie, Göttingen, Germany

ErbB2, a transmembrane tyrosine kinase, forms both homo- and heterodimers with other members of the erbB family. Previously we found evidence for the existence of membrane regions in breast cancer cells where the homoassociation of erbB2 is anomalously high. In our current work we set out to characterize the dimensions and the possible physiological importance of these regions. We used a scanning near-field optical microscope to visualize erbB2 clustering in fluorescent antibody-labeled cells. Quiescent MDA453 and SKBR3 breast tumor cells showed a conspicuously uneven distribution of erbB2 with a cluster diameter of 450 nm. A single cluster contains about 1000-2000 erbB2 proteins in unstimulated SKBR3 cells. To correlate the activation state of erbB2 with the diameter of clusters, we used three different agents to stimulate SKBR3 cells: EGF and heregulin treatment, and incubation at 37 oC with the 4D5 antibody known to be a partial agonist. Each of these treatments increased the size of erbB2 clusters, although they exert their activation effect differently. The EGF-induced effect could be blocked with an EGF-receptor specific receptor tyrosine kinase inhibitor. Our results imply that erbB2 is concentrated in protein clusters in an activation dependent manner. In addition to the acknowledged short scale association of erbB proteins (e.g. dimers), the clustering of several hundreds of proteins may also be significant in signal transduction by ensuring a high local concentration of signaling proteins.
This work was supported by OTKA research grants F020102, F022725, T019372 from the Hungarian Academy of Sciences. AJ was supported by a long-term EMBO fellowship.