Activation-dependent clustering of the erbB2 receptor tyrosine kinase detected by scanning near-field optical microscopy

ErbB2 (HER2, Neu), a member of the epidermal growth factor (EGF) receptor t yrosine kinase family, is often overexpressed in breast cancer and other ma lignancies, ErbB2 homodimerizes but also presents as a common auxiliary sub unit of the EGF and heregulin receptors (erbB1 or EGFR; and erbB3-4, respec tively), with which it heteroassociates, ErbB2 is generally regarded as an orphan (ligand-less) receptor with a very potent kinase domain activated ei ther via its associated partners or constitutively as a consequence of disc rete mutations. It follows that the extent and regulation of its cell surfa ce interactions are of central importance. We have studied the large-scale association pattern of erbB2 in quiescent and activated cells labeled with fluorescent anti-erbB2 monoclonal antibodies using scanning near-field opti cal microscopy (SNOM), ErbB2 was found to be concentrated in irregular memb rane patches with a mean diameter of approx. 0.5 mu m in nonactivated SKBR3 and MDA453 human breast tumor cells. The average number of erbB2 proteins in a single cluster on nonactivated SKBR3 cells was about 10(3). Activation of SKBR3 cells with EGF, heregulin as well as a partially agonistic anti-e rbB2 monoclonal antibody led to an increase in the mean cluster diameter to 0.6-0.9 mu m, irrespective of the ligand, The EGF-induced increase in the erbB2 cluster size was inhibited by the EGFR-specific tyrosine kinase inhib itor PD153035, The average size of erbB2 clusters on the erbB2-transfected line of CHO cells (CB2) was similar to that of activated SKBR3 cells, a fin ding correlated with the increased base-line tyrosine phosphorylation of er bB2 in cells expressing only erbB2, We conclude that an increase in cluster size may constitute a general phenomenon in the activation of erbB2.