Interactions Between Insulin and Its Receptors After the Initial Binding Event: Functional Heterogeneity and Relationships to Insulin Degradation

Abstract

We studied the rate at which insulin dissociates from adipocytes and the ability of these cells to degrade insulin as a function of the length of exposure to the hormone. As the length of the association phase increases, the overall rate of insulin dissociation decreases. After 6 min of association at 24°C, the t1/2 of dissociation is 8 min and after 90 min of association the t1/2 of dissociation is 32 min. This time-dependent slowing of dissociation is progressive until steady-state binding conditions are reached during the association phase; after this point (90 min at 24°C), no further slowing of dissociation occurs. Comparable results were seen at 37°C, and at either temperature essentially 100% of the bound insulin eventually dissociates from the cells. Thus, no tendency for irreversible binding was observed. Similar results were obtained with isolated plasma membranes, and a variety of inhibitors of cell functions failed to influence the time-dependent slowing of dissociation. This suggests that the processes responsible for the changes in dissociation rate are independent of intracellular factors and are inherent in the plasma membrane. The results are compatible with the concept that insulin initially binds to a low affinity (fast dissociating) receptor and that a minority of these complexes are then converted to a high affinity (slow dissociating) state by a time-dependent process. The relationship of the above phenomenon to insulin degradation was examined by assessing the chemical nature of the radioactive material bound to the cells at different times of association and by measuring dissociation of both intact and degraded insulin. The results indicate that only intact insulin dissociates from the low affinity (fast dissociating) state of the receptor. In contrast, once the high affinity complexes are formed, both intact and degraded material can slowly dissociate. Insulin bound to the high affinity (slow dissociating) state of the receptor is a direct and efficient substrate for the insulin-degrading system. However, when insulin degradation was completely blocked by the sulfhydrylagent n-ethyl maleimide, the time-dependent slowing of dissociation of intact insulin was unaffected. Thus, development of the slow-dissociating state of the insulin receptor complex mediates cellular insulin degradation, but the process of insulin degradation is not necessary for the development of the slow-dissociating state of the insulin receptor complex. In conclusion: (a) A time-dependent slowing of the overall insulin dissociation rate was observed as the length of the association phase increased; however, no tendency toward irreversible binding of insulin to adipocytes was observed under any conditions, (b) This phenomenon is independent of a number of intracellular processes studied and is inherent in the plasma membrane, (c) The receptors to which insulin is bound early in the association phase do not degrade insulin, and, in fact, they protect the hormone from destruction, whereas a portion of the receptors to which insulin is bound late in the association phase mediate insulin degradation, (d) The data are consistent with the concept that insulin initially binds to a low affinity cell surface receptor and that some portions of these complexes are converted to a high affinity form by a time-dependent plasma membrane-associated process. The low affinity receptors have a rapid dissociation rate, are of high capacity, and do not degrade insulin. The high affinity receptors have a slow dissociation rate, are of low capacity, and mediate insulin degradation.