Kinetics of insulin receptor transit to the removal from the plasma membrane. Effect of insulin-induced down-regulation in 3T3-L1 adipocytes

The heavy isotope density shift method, in combination with a procedure for labeling cell surface insulin receptors, was used to determine the rate of transit of receptor to the cell surface from their site of synthesis and to follow the net rate of receptor removal from the plasma membrane in 3T3-L1 adipocytes. To label surface receptors, ¹²⁵I-insulin was bound to cells at 4 °C and then covalently cross-linked to the receptors with disuccinimidyl suberate. The identity of the surface-labeled product as insulin receptor was established by immunoprecipitation with antireceptor antibody and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fully differentiated 3T3-L1 adipocytes were shifted to medium containing heavy (>95% ¹⁵N, ¹³C and ²H) amino acids. The rates of appearance of newly synthesized heavy receptor at the cell surface and the loss of previously synthesized light receptor from the cell surface were followed by resolving labeled heavy and light surface receptors in CsCl density gradients and quantitating labeled receptor subunits by gel electrophoresis. It was shown that 2.5-3.0 h are required for newly synthesized insulin receptor to reach and become functional in the plasma membrane. Insulin-induced down-regulation of cellular insulin receptor level had no effect on the time required for the newly synthesized receptors to reach the cell surface. Down-regulation, however, increased the first order rate constants for the inactivation of cell surface insulin receptors from 0.046 to 0.10 h^-1. The fact that the rate constants for inactivation of cell surface and total cellular insulin receptors were identical in the up-regulated state (0.046 and 0.044 h^-1, respectively) or in the down-regulated state (0.10 an 0.096 h^-1, respectively) suggests that the rate-limiting step in the receptor inactivation pathway occurs at the cell surface.