Expression of functional insulin-like growth factor-I receptors by human amnion cells

Abstract

The purpose of the study was to investigate whether amnion cells contain functional insulin-like growth factor-I receptors. To test whether human amnion cells contain insulin-like growth factor-I receptors, radioligand binding studies, affinity cross-linking studies, and Northern blot analysis were conducted in primary amnion cells and in an immortal amnion cell line (WISH). To test whether the insulin-like growth factor-I receptors on amnion cells are functional, cytochalasin B-inhibitable 2-deoxyglucose uptake was measured after stimulating the cells with insulin-like growth factor-I. Radioligand binding studies demonstrated that primary amnion cells and WISH cells contained a single class of high-affinity receptors with an apparent dissociation constant of 0.18 +/- 0.04 nmol/L and a receptor concentration of 79 +/- 26.2 fmol/mg protein and dissociation constant of 0.44 +/- 0.03 nmol/L and a receptor concentration of 33.3 +/- 6.45 fmol per 106 cells, respectively. Affinity cross-linking studies revealed two major insulin-like growth factor-I binding sites, 135 and 270 kd. Both primary amnion cells and WISH cells exhibited cytochalasin B-inhibitable tritiated 2-deoxyglucose uptake in response to insulin-like growth factor-I treatment. Finally, treatment of WISH cells caused tyrosine phosphorylation of three proteins (molecular weight, 116, 95.4, and 83.5 kd) was observed by Western blotting with antiphosphotyrosine antibodies. These results provide the first evidence that human amnion epithelial cells contain functional high-affinity insulin-like growth factor-I receptors that mediate glucose transport.