Insulin-like Growth Factor II Binding to Cultured Human Chondrosarcoma Cells

Abstract

Abstract

Cultured cells originally derived from a human chondrosarcoma (A1684) were used to investigate somatomedin binding in terms of kinetics and specificity. In this study, the rat somatomedin, multiplication-stimulation activity (MSA) was utilized. While the human chondrosarcoma cells did not exhibit a mitogenic response to MSA, the rate of transport of glucose and amino acids was significantly increased. In competitive binding experiments a specific insulin-insensitive MSA receptor was identified which showed half maximal displacement of tracer at a concentration of 250 ng/ml of MSA using whole cells. This receptor had an affinity constant of 4.8 × 10⁷ M ⁻¹. Kinetic analysis of MSA binding to membrane preparations and to Triton X-100 solubilized membranes revealed an increase in the binding affinity to 1.28 × 10⁸ M ⁻¹ and 2.8 × 10⁸ M ⁻¹, respectively. Of particular significance is the observation that these cells have especially high levels of MSA receptors. Determination of binding capacity revealed that these cells contain approximately 1.9 × 10⁶ MSA receptors per cell and therefore are an excellent model system for the characterization and purification of somatomedin receptors. Affinity labeling of the MSA receptor using the chemical crosslinking reagent, disuccinimidyl suberate, confirmed that this receptor was of the type II class of somatomedin receptors and exhibited a molecular weight of 218,000 under nonreducing conditions.