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J. Biol. Chem., Vol. 263, Issue 32, 16561-16567, 11, 1988

Calcium influx: an intracellular message of the mitogenic action of insulin-like growth factor-I

I Kojima, H Matsunaga, K Kurokawa, E Ogata and I Nishimoto
Fourth Department of Internal Medicine, University of Tokyo School of Medicine, Japan.

When G0-arrested BALB/c 3T3 cells were treated sequentially with platelet-derived growth factor and epidermal growth factor, cells became responsive to insulin-like growth factor-I (IGF-I). In these primed competent cells, 1 nM IGF-I elicited an approximately 3-fold increase in the calcium influx rate. IGF-I-induced calcium influx was relatively slow in onset and continued for at least 2 h in the presence of IGF-I. When a single Ca2+ channel current was studied by the patch- clamp technique using the cell-attached mode, inward currents with unitary conductance of 19 pS were observed in the presence of 1 nM IGF- I in the patch pipette. IGF-I-sensitive inward current was independent of membrane potential and was activated by a high concentration of insulin. Accordingly, 1 nM IGF-I caused a gradual increase in cytoplasmic free calcium concentration measured by fura2. The action of IGF-I on calcium influx was dependent on extracellular calcium, and IGF- I did not stimulate calcium influx when extracellular calcium concentration was reduced to 10 microM. Both cobalt and tetramethrin blocked the action of IGF-I on calcium influx without affecting the binding of 125I-IGF-I. In primed competent cells, IGF-I-stimulated [3H]thymidine incorporation was dependent on extracellular calcium and was attenuated by cobalt and tetramethrin. When cell-bound 125I-IGF-I was cross-linked by use of disuccinimidyl suberate, a 130-kDa protein was radiolabeled. Affinity labeling of the 130-kDa protein, presumably the alpha-subunit of the IGF-I receptor, was blocked by excess amount of unlabeled IGF-I. These results suggest that relatively low concentrations of IGF-I stimulate calcium influx in primed competent BALB/c 3T3 cells by activating a calcium-permeable cation channel via the IGF-I receptor and that calcium influx may be a critical intracellular message of the progression activity of IGF-I.
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