Abstract
The mechanism of insulin-induced down-regulation of surface membrane insulin receptors was studied in the muscle cell line BC3H-1. Down-regulation for the differentiated myocytes is dose- and time-dependent with a half-maximum response at 0.5 nM insulin and a maximum decrease of 50% in the number of surface insulin receptors following exposure to 20 nM insulin for 18 h at 37 degrees C, as confirmed by Scatchard analysis. These receptors were fully recoverable upon lysis of the down-regulated myocyte with Triton X-100, demonstrating that down-regulation is mediated solely by insulin-induced receptor internalization without detectable receptor degradation. Phospholipase C treatment of intact down-regulated cells and Triton X-100 treatment after subcellular fractionation showed that no cryptic or masked receptors were detectable within the plasma membrane. Insulin-induced receptor internalization was dependent upon cellular energy production, protein synthesis, and endocytosis, but was insensitive to agents which primarily affect lysosomal, cytoskeletal, or transglutaminase activities. The magnitude of insulin-induced down-regulation and the kinetics of down-regulation and recovery of cell surface receptors indicate that the surface and internal receptor pools are in dynamic equilibrium with each other. The kinetic data are accommodated by separate internalization rate constants for the unoccupied (0.01 h-1) and occupied (0.11 h-1) surface receptors and a single recycling rate constant (0.11 h-1) for the internalized receptors. This model also explains the previous apparently paradoxical finding in several other systems that down-regulation is more sensitive to hormone than hormone-receptor binding under physiologic conditions. Down-regulation in BC3H-1 myocytes, therefore, appears to be mediated solely by an insulin-induced increase in the receptor internalization rate constant and a consequent shift in the dynamic equilibrium between the surface and internalized receptor pools, resulting in a 50% decrease in the number of cell surface receptors. In other systems where the internalized hormone receptor is a substrate for rapid degradation, the essential role of this shift in mediating the down-regulation process may be obscured.
Highlights
From the Departments of Medicine and Biochemistr-y., Universityof South Florida and Veterans’Administration Medical Centers, Tampa, Florida33612
The mechanism of insulin-induced down-regulation As the first step in the mechaniosfmpolypeptide hormone of surface membane insulin receptors was studied in action, hormone binding tospecific plasma membrane recepthe muscle cell line BC3H-1
Down-regulation of insulin receptors in thadeipocyte is apparenot nly whenrecycling of insulin occupied (0.01 h-l) and occupied (0.11 h-’) surface receptors to the cell surface is inhibited with Tris [11, 12]
Summary
Vol 259, No 4, Issue of February 25, pp. 23462354,1984 Printed in U.S.A. From the Departments of Medicine and Biochemistr-y., Universityof South Florida and Veterans’Administration Medical Centers, Tampa, Florida33612. On the basis of these studies, we traction of nonspecific binding as determined by the addition of 10 propose a model for insulin receptor down-regulation involving a dynamic equilibrium between surface and intracellular pools of insulinreceptors which is controlled by therate constants for receptor internalization and recycling. The insulin receptor appears to be relatively insensitive to direct heterotropic regulation by unrelated hormones, suggesting that thein vivo insulin resistance associated with elevated levels of steroid and other hormones is largely mediated by postreceptor effects [38,39,40,41]
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