Journal of Clinical Endocrinology & Metabolism, Vol 58, 535-543, Copyright © 1984 by Endocrine Society

ARTICLES

Affinity-labeled somatomedin-C receptors and binding proteins from the human fetus

JD Grizzard, AJ D'Ercole, JR Wilkins, BM Moats-Staats and RW Williams

We investigated the ontogeny of the human placental membrane somatomedin-C (Sm-C)/insulin-like growth factor I (IGF-I) receptor by affinity labeling with the cross-linking agent disuccinimidyl suberate (DSS). Specific Sm-C receptors, identified from as early as 6 weeks gestation, demonstrated no apparent structural changes through the course of gestation. Second trimester human fetal brain membranes cross- linked to [125I]Sm-C exhibited an identical pattern of receptor binding. These findings in both placenta and fetal brain membrane are consistent with the proposed heterotetrameric structure of the Sm-C/IGF- I receptor. Using a similar DSS cross-linking technique, we identified Sm-C-binding proteins in second trimester amniotic fluid [apparent molecular mass (Mr), 38,000 and 35,000] and term cord plasma (Mr, 41,000, 38,000, and 35,000). Identically sized binding components (Mr, 35,000-45,000) were also found in membrane preparations of preterm placenta and brain after cross-linking. Evidence that these binding species represent contamination of preterm membrane preparations with soluble amniotic fluid and/or fetal plasma Sm-C-binding proteins and that they are not derived from membrane receptors is as follows: (1) these binding species, like amniotic fluid and cord plasma binding proteins, were insensitive to competition with insulin (in concentrations as high as 0.6 mg/ml), a characteristic not shared with membrane receptor binding components; (2) these binding species were largely removed from placental membranes by extensive washing and appeared in the supernate when membrane preparations were incubated at 4 C for 18 h, indicating that they were soluble and not an integral part of the particulate membrane; (3) limited proteolysis of placental membrane preparations did not result in the appearance of similar binding species; and (4) preparation of preterm placental membranes in the presence of protease inhibitors did not eliminate these binding species. Use of traditional methodology to study binding of somatomedin to receptors in membranes prepared from preterm human tissues may be misleading because of contamination by amniotic fluid and/or plasma- derived binding proteins.