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

OBJECTIVES: The purpose of the study was to investigate whether amnion cells contain functional insulin-like growth factor-I receptors. STUDY DESIGN: 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. RESULTS: 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. CONCLUSION: 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.