Characterization of the Somatomedin-C/Insulin-like growth factor I (SM-C/IGF-I) receptor on cultured human fibroblast monolayers: Regulation of receptor concentrations by SM-C/IGF-I and Insulin

We have characterized ¹²⁵I-labeled somatomedin-C/insulin-like growth factor 1 ([¹²⁵I]SM-C/IGF-I) binding to cultured human fibroblast monolayers and have investigated the modulation of receptor concentrations by SM-C/IGF-I and insulin. Specific binding of [¹²⁵I]SM-C/IGF-I to confluent monolayers at 15 C reached a steady state in 2.5 h and averaged 11.37 ± 0.71% (mean ± SEM)/1.5 × 10⁶ cells. At 30 C, binding occurred more rapidly, peaking at 30 min, but averaged only 4%. Binding was reversible at both temperatures, and degradation of radioligand by fibroblast monolayers was negligible. Half-maximal inhibition of [¹²⁵I]SM-C/IGF-I binding was observed at SM-C/IGF-I concentrations of 7 ng/ml. Compared to SM-C/IGF-I, the relative potencies of other hormones was: IGF-II, 1:10; multiplication-stimulating activity, 1:17, porcine insulin, 1:300; porcine proinsulin, 1:1000; and human GH nonreactive. Scatchard analysis was consistent with a single class of receptors, with a K^a of 1.07 × 10⁹ M⁻¹ and 20,000 sites/cell. Preincubation of fibroblast monolayers with either SM-C/IGF-I or insulin resulted in a time- and concentration-dependent reduction in the binding of [¹²⁵I]SM-C/IGF-I. Twenty-hour preexposure of monolayers to 2 × 10⁻⁸ M SM-C/IGF-I induced a 50% decrease in the binding of [¹²⁵I]SM-C/IGF-I, while preincubation with 10⁻⁷ M SM-C/IGF-I resulted in a 70% decrease in binding. Similarly, binding was reduced 34% and 45% after preincubation with 10⁻⁷ or 10⁻⁶ M insulin, respectively. Half-maximal reduction in binding required preincubation for 10 h at 37 C, and maximal decreases in binding occurred after 20 h of preincubation with either SM-C/IGF-I or insulin. These studies thus demonstrate that: 1) cultured human fibroblasts possess highly specific SM-C/IGF-I receptors which may be studied directly in cell monolayers, and 2) SM-C/IGF-I receptor concentrations are directly modulated by ambient concentrations of either SM-C/IGF-I or insulin, thus emphasizing the structural and functional homology of these peptides.