Immunoelectron microscope analysis of epidermal growth factor receptor (EGFR) in isolated Mytilus galloprovincialis (Lam.) digestive gland cells: Evidence for ligand-induced changes in EGFR intracellular distribution

In mammalian cells, the binding of epidermal growth factor (EGF) to its rec eptor (EGFR), a glycoprotein with intrinsic tyrosine kinase activity, leads to the pleiotropic responses to EGF. Among these, a negative feedback resp onse by stimulation of receptor internalization and lysosomal degradation, this attenuating signal transduction. In this work, data are reported on the identification of specific EGFRs in isolated digestive gland cells from the marine mussel (Mytilus galloprovinc ialis Lam.) by immunoelectron microscopy. In control digestive cells, EGFR immunoreactivity was mainly associated with cytoplasmic membrane structures and, to a lesser extent, the cell membrane. The presence of EGFR-like rece ptors was confirmed by Western blotting of digestive gland cell extracts wi th two different monoclonal antibodies that recognize either intracellular or extracellular epitopes. The addition of mammalian EGF resulted in significant time and temperature- dependent changes in EGFR subcellular distribution in mussel cells. In cell s exposed to EGF for 0-15 min at 4 degrees C, the distribution of EGFR was not significantly different from that of the control cells. On the other ha nd, at 18 degrees C, an increased labelling along the cell membrane was obs erved after 5-10 min after EGF addition, with a concomitant decrease in the cytoplasmic signal. Moreover, after 20 min of exposure to EGF, ligand bind ing apparently resulted in EGFR compartmentation within the lysosomes. Thes e observations were confirmed by quantitative analysis of EGFR labelling at different times of EGF exposure. Similar results were obtained utilizing t he two different monoclonal antibodies. The results indicate that, in musse l digestive cells, the binding of heterologous EGF to specific receptors in duces a negative feedback response by stimulating the lysosomal degradation of EGFR, thus suggesting the presence of mechanisms responsible for recept or downregulation similar to those observed in mammalian cells. J. Exp. Zoo l. 286:690-698, 2000. (C) 2000 Wiley-Liss, Inc.