Pertussis Toxin Sensitive G-proteins Are Not Involved in the Mitogenic Signaling Pathway of Insulin-like Growth Factor-I in Normal Rat Kidney Epithelial (NRKE) Cells

There is controversy as to whether or not a pertussis toxin sensitive G-protein is involved in the signaling pathway of insulin-like growth factor-I. We have used normal rat kidney epithelial (NRKE) cells to ask whether or not a pertussis toxin sensitive G-protein was involved in IGF-I stimulated DNA synthesis. NRKE cells express both IGF and IGF-II/M6P receptors and respond to IGF-I with increased thymidine incorporation into DNA. Under many circumstances incubation of cells/cell membranes with GTP analogues will inhibit binding of ligands that are linked to a G-protein-receptor pathway. However, when NRKE membrane preparations were incubated with 125I-IGF-I or 125I-IGF-II in the presence or absence of GTP gamma S, ATP and GTP, binding of the radioligands was not affected by the GTP-analogue. IGF-I and factors from serum of hypophysectomized rats (HRS) stimulated [3H]thymidine incorporation into DNA of NRKE cells. Under serum-free conditions in the presence of EGF (2 ng/ml) and PDGF (1 ng/ml) pertussis toxin over a wide range of doses had no effect upon IGF-I stimulated [3H]thymidine incorporation into DNA of NRKE cells. In addition, PT at a dose of 100 ng/ml had no effect on IGF-I(0.2-50 ng/ml) stimulated DNA synthesis of NRKE cells. However, PT at doses of 5, 50, 500, 5000 and 50,000 ng/ml was capable to ADP-ribosylate a 40 kDa protein in NRKE cell plasma membrane preparations corresponding to known PT-sensitive G-proteins. We conclude, that (1) PT-sensitive G-proteins and both IGF-I and IGF-II/M6P receptors are present in NRKE cell plasma membrane preparations, and most importantly, that (2) PT-sensitive G-proteins are not involved in the mitogenic signaling pathway of IGF-I in NRKE cells.