Identification and Characterization of 125I-Insulin-Like Growth Factor-II Binding Sites on the Muscular Layer of Stem Villi Vessels of Human Term Placenta1

Abstract

The primary function of the placenta is to ensure an optimal environment for fetal growth and development. In normal pregnancy, placental vascular tone regulation assures fetus well-being and normal development by maintaining adequate blood flow so as to ensure materno-fetal exchanges. In human placenta, synthesis of insulin-like growth factor (IGF)-II and specific binding sites have been previously characterized in the trophoblast; in contrast, no studies have dealt with this subject in the fetoplacental vascular system, particularly in stem villi vessels. We thus investigated whether membranes of the muscular layer of stem villi vessels contained 125I-IGF-II binding sites. Two complementary approaches were used: 125I-IGF-II binding and affinity cross-linking studies. 125I-IGF-II labeled, in a saturable and noncooperative manner, a single class of high-affinity binding sites characterized by a Kd of 1.24 +/- 0.26 nM (n = 6), a maximum binding capacity (Bmax) of 3.02 +/- 0.45 pmol/mg protein, and a Hill coefficient of 1.00 +/- 0.15. Competitors for 125I-IGF-II binding to membranes were in the order of potency IGF-II > IGF-I. Insulin was not a competitor. Affinity cross-linking of membranes with 125I-IGF-II, followed by SDS-PAGE and autoradiography, revealed two labeled bands: a protein complex of 250 kDa, which corresponds to the type II IGF receptor, and another of 135 kDa, corresponding to the type I IGF receptor. Only IGF-II could displace 125I-IGF-II binding from the major 250-kDa band, while 125I-IGF-II bound to the minor 135-kDa band was displaced by either IGF-I, IGF-II, or insulin. In conclusion, high levels of specific binding sites for 125I-IGF-II are present in the muscular layer of stem villi vessels, which are considered placenta resistance vessels. The involvement of both type I and type II IGF receptors in the growth-promoting action of IGF-II remains to be determined in the fetoplacental vascular system.

Differential Distribution of Binding Sites for 125I-Insulin-Like Growth Factor II on Trophoblast Membranes of Human Term Placenta

“…The maximal binding of 125 1-IGF-II to the basal plasma membranes is similar to that found earlier for the muscle layer of the stem villi vessels of the human term placenta [19], but significantly (p < 0.0001) smaller than that for the microvillous membranes. These findings are corroborated by cross-linking experiments showing that the total 12 5 I-IGF-II bound to microvillous membranes is 1.5 times that of 1 25 I-IGF-II bound to basal plasma membranes.…”

“…The optimal experimental conditions for the binding of 1 25 I-IGF-II to microvillous and basal plasma membranes were determined as previously described [19]. The specific binding of 125 I-IGF-II to both the trophoblastic membranes was time and temperature dependant, and increased linearly with the protein concentration over the range 5-25 pxg for the microvillous membrane and 5-30 ,ug for the basal plasma membrane.…”

“…Recent studies have described the genes for IGF-II-and IGF-binding proteins (IGF-BP) in the developing human placenta and amniotic fetal membranes from very early gestation (6 wk) to term [7]. We have also described IGF-II-binding sites on the muscular layer of the stem villi vessels of the human term placenta, which are physiologically important since they act as placental resistance vessels [19]. The present study describes the IGF-IIbinding sites in the trophoblast of the human term placenta and compares their binding characteristics and their distribution in the brush-border microvilli and basal plasma membranes, using a pharmacological approach and crosslinking experiments.…”