Characterization of somatomedin/insulin-like growth factor receptors and correlation with biologic action in cultured neonatal rat astroglial cells

Abstract

The role of somatomedin/insulin-like growth factors (Sm/IGFs) in neural growth and development is not clearly defined. To characterize Sm/IGF receptors and to correlate binding with the biologic actions of Sm/IGFs in a homogeneous population of neural cells, we isolated and studied a nearly pure population of cultured astroglial monolayers derived from cerebral cortices of neonatal rats. Binding of radiolabeled Sm/IGFs was specific, saturable, and reversible, with 90% of the binding occurring within 6 hr of incubation at 4 degrees C. Competitive binding studies with Sm-C/IGF I yielded curvilinear Scatchard plots, while studies with IGF II and multiplication stimulating activity (MSA) yielded linear plots, suggesting that 125I-Sm-C/IGF I binds to more than 1 receptor species, and 125I-IGF II and 125I-MSA bind to one only. These findings were supported by affinity-labeling studies with radiolabeled Sm/IGFs using disuccinimidyl suberate as a cross-linking agent. Sm-C/IGF I appeared to bind to both type I and II Sm/IGF receptors, because cross-linked 125I-Sm-C/IGF I-receptor complexes with molecular weight (Mr) of greater than 300,000 (300K) and 130K (type I receptor) were observed under nonreducing and reducing conditions, respectively, as were 220 and 260K complexes (type II receptor) under the same respective conditions. 125I-IGF II and 125I-MSA, however, bound only to the Mr 220 and 260K moieties under nonreducing and reducing conditions, respectively, suggesting that these peptides bind only to the type II receptor. Competitive binding studies of the cross-linked moieties were consistent with this interpretation. In contrast, 125I-insulin bound poorly to astroglia (less than 0.5% specific binding), and cross-linking studies could not definitely distinguish among low-affinity binding to the type I Sm/IGF receptor, binding to a paucity of insulin receptors, or both. In addition, by combining autoradiography to localize 125I-Sm/IGFs binding on astroglial cells and immunocytochemistry with anti-glial fibrillary acidic protein to identify the cell type, we have demonstrated cell-surface binding and apparent internalization of radiolabeled Sm/IGFs. Concurrent studies of Sm/IGF stimulation of 3H-thymidine incorporation revealed that these cells were most sensitive to Sm-C/IGF I, followed by IGF II and MSA, and insulin. MSA and IGF II, however, were the most potent followed by Sm-C/IGF I and then insulin. Half-maximal stimulations of 3H-thymidine incorporation corresponded closely with half-maximal binding displacement for Sm-C/IGF I and less so for IGF II and MSA.(ABSTRACT TRUNCATED AT 400 WORDS)