Abstract
Epidermal growth factor (EGF) stimulates the turnover of phosphoinositides in A431 cells. In cells that were pretreated with EGF for 30 min at 37 degrees C and then washed to remove surface-bound hormone, a 70-100% decrease in the EGF-stimulated production of inositol monophosphate, inositol bisphosphate, and inositol triphosphate was noted when the cells were exposed to the agonist a second time. Since only a 15% decrease in receptor number was observed in these pretreated cells, the loss of responsiveness to EGF for the production of inositol phosphates could not be attributed to a down-regulation of the EGF receptors. These data suggest that pretreatment of A431 cells with high concentrations of EGF leads to a desensitization of the EGF receptor. This desensitization of the receptor by EGF is apparent within 10-15 min of the addition of EGF and is maximal by 30 min. The desensitization appears to be homologous in nature since pretreatment of cells with EGF did not diminish their responsiveness to bradykinin; and conversely, pretreatment with bradykinin did not diminish the subsequent responsiveness of the cells to EGF. Desensitization to EGF was observed in cells in which protein kinase C had been down-regulated by prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate, implying that EGF receptor desensitization is independent of protein kinase C. The desensitizing effects of EGF on growth factor-induced phosphatidylinositol turnover could be prevented by pretreatment of the cells with the calmodulin antagonist trifluoperazine, suggesting that calmodulin may be involved in the regulation of EGF receptor sensitivity.
Highlights
The mechanisms of receptor desensitization have been extensively studied for the adenylate cyclase-coupledP-adrenergic receptor.Heterologous desensitization of this receptor is associated with its phosphorylation by CAMP-dependent protein kinase [2, 3]
To determinewhether thisapparent desensitization was limited to effects on ligand internalization or represented a more generalized change in the responsiveness of the Epidermal growth factor (EGF) receptor, we examined the effects of prolonged preincubation with EGF on the ability of the hormone to subsequently stimulate PI turnover inA431 cells
PI Turnover cells with EGF decreases theextent of internalization of subsequently bound lZ5I-EGFT. hese findings were interpreted as being the result of a desensitization of the EGF receptor
Summary
Materials-EGF was purified from submaxillaryglands by the method of Savage and Cohen [23]. my~-[~H]Inositowlas obtained. A t 37 "C to induce desensitization; (ii) washing of the mono- stimulated inositol phosphate production following a second layer with HBSS and an acid buffer at 4 "C to remove un- challenge with the growth factor (datanot shown) This bound and surface-bound EGF; (iii) treatment of the cells indicates that elevated levels of inositol phosphates do notby with or without EGF for 20 min at 37 "C to stimulate PI themselves cause a reduction of EGF-stimulated PI turnover. When cells that had been treated with TFP were subfor receptor internalization or if they affected other EGF- jected to the desensitization regimen, basal levels of inositol induced responses, the effect of the inhibitor TFP on desen- phosphates were elevated as usual; the cells still sitization of EGF-stimulated PI turnover was measured.
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