Transmembrane signalling at epidermal growth factor receptors overexpressed in NIH 3T3 cells. Phosphoinositide hydrolysis, cytosolic Ca2+ increase and alkalinization correlate with epidermal-growth-factor-induced cell proliferation

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Abstract
NIH 3T3 cells, which express a small number of EGF (epidermal growth factor) receptors, are poorly responsive to EGF. However, when the same cells overexpress the cloned human EGF receptor (EGFR T17 cells), they display EGF-dependent transformation. In EGFR T17 cells (but not in the parental NIH 3T3 cells), EGF is shown here to trigger polyphosphoinositide hydrolysis as well as the generation of the ensuing intracellular signals, the increase in the cytosolic Ca2+ concentration ([Ca2+]i) and pH. EGF induced a large accumulation of inositol 1,4,5-trisphosphate, with a peak at 15-30 s and a slow decline thereafter. Other inositol phosphates (1,3,4-trisphosphate and 1,3,4,5-tetrakisphosphate) increased less rapidly and to a lesser degree. [Ca2+]i increased after a short lag, reached a peak at 25 s and remained elevated for several minutes. By use of incubation media with and without Ca2+, the initial phase of the EGF-induced [Ca2+]i increase was shown to be due largely to Ca2+ release from intracellular stores. In contrast with previous observations in human A431 cells, the concentration-dependence of the EGF-triggered [Ca2+]i increase in EGFR TR17 cells paralleled that of [3H]thymidine incorporation. It is concluded that polyphosphoinositide hydrolysis, [Ca2+]i increase and cytoplasmic alkalinization are part of a spectrum of intracellular signals generated by the activation of one single EGF receptor type. These processes might be triggered by the receptor via activation of the intrinsic tyrosine kinase activity. Large stimulation of DNA synthesis and proliferation by EGF in EGFR T17 cells could be due to a synergistic interplay between the two signal pathways initiated by tyrosine phosphorylation and polyphosphoinositide hydrolysis.