| Description |
Amplification of the Epidermal Growth Factor Receptor (EGF-R) is closely associated with oncogenesis. We attempted to elucidate the mechanism used by receptor amplified cells to increase EGF-R signaling levels and/or duration. Constitutive EGF-R associated phosphotyrosine (PY), on a per cell basis, increases with receptor amplification. Moreover, PY levels appear to increase linearly with receptor level. We found that the simultaneous application of the EGF-R antagonistic antibody 225 and metalloprotease inhibitors, which block ligand production, resulted in a synergistic attenuation of this PY. Therefore, most, if not all, of the constitutive PY is ligand dependent. However, by measuring the concentration of Transforming Growth Factor Alpha (TGF?) secreted into the media, we showed that this ligand dependent increase in constitutive PY was not due to an increase in autocrine ligand expression. Furthermore, we found that cell lines can capture most of the autocrine ligand they produce regardless of receptor expression level. Using a modified pulse-chase assay, we determined that ligand was recaptured more efficiently following recycling in cells displaying receptor amplification. This increased capture efficiency at least partially explains the increased level of constitutive PY observed following receptor amplification. In another series of experiments, we found that the regulatory apparatus of cells with receptor amplification was more than adequate to handle the ligand levels those cells would see under physiological conditions. Therefore, a limited capacity of these regulatory systems is unlikely to account for the increased constitutive PY. However, PY was removed faster than EGF-R protein was degraded in cells with low receptor numbers while PY is removed at a rate similar to receptor degradation in amplified cells. We found that the number of intracellular EGF-Rs activated was larger than the number of available ligand molecules in the amplified lines. This result indicates a degree of signal amplification. However, this effect is not observed in the low receptor number expressing cell lines. We propose that unoccupied, intracellular EGF-R contribute to the increased receptor activity we observe in receptor amplified cell lines by a "ligand echo" effect. |
