Perturbation of the Insulin Receptor of Isolated Fat Cells with Proteolytic Enzymes DIRECT MEASUREMENT OF INSULIN-RECEPTOR INTERACTIONS

Abstract The effects of proteolytic digestion of isolated adipose tissue cells have been studied by simultaneously examining the metabolic and receptor-binding consequences of tryptic digestion. Insulin-receptor interactions in the intact cell are measured directly and independently of the metabolic processes of the cell by determining the specific binding of 125I-insulin to cells. Tryptic digestion of fat cells leads to a selective and profound fall in the affinity of the receptor for insulin. This occurs under conditions where the maximal insulin response (glucose oxidation) and the total amount of receptor are unaffected. These effects are easily detected by either measuring glucose oxidation or insulin binding as a function of increasing concentrations of insulin. More drastic tryptic digestion results in additional and qualitatively different effects. Such digestion either destroys more critical regions of the receptor or prevents the normal transmission of signals from the insulin-receptor complex to the glucose transport mechanisms of the cell membrane. Effects similar to those observed with trypsin are obtained by digesting fat cells with α-chymotrypsin. Agarose derivatives containing covalently linked trypsin produce the same effects that are observed with trypsin in solution. This is strong evidence that the effects of tryptic digestion result exclusively from perturbations of superficial structures of the cell membrane. The decreased affinity of the insulin receptor resulting from tryptic digestion does not change during prolonged periods of incubation. There is no evidence that this specific and well characterized effect of tryptic digestion is subject to facile and spontaneous repair. Changes in the basal and insulin-stimulated rates of glucose oxidation are observed during incubation, but these changes are complicated and not easily interpreted. Digestion of fat cells with phospholipase C or phospholipase A increases quite appreciably the total amount of insulin receptor which is exposed to the medium. The insulin receptor in such cells becomes much more susceptible to tryptic digestion. In these cells trypsin cleaves regions more critical to the binding function of the receptor since severe destruction of the receptor occurs without significant effects on affinity. No displacement of specific binding of 125I-insulin to fat cells is observed with high concentrations of several enzymatically inactive precursors and derivatives of proteolytic enzymes. Trypsin and α-chymotrypsin (at 4°) do not cause displacement of 125I-insulin binding. These enzymes probably cannot specifically interact with the same receptor as insulin in the fat cell. The insulin receptor of the fat cell is normally located in the membrane in such a way that "peripheral" glycopeptide regions are exposed to the solvent and are susceptible to digestion by proteolytic enzymes and neuraminidases. Cleavage of certain parts of these glycopeptides alters but does not abolish the function of the receptor. The more critical and "central" regions of the receptor structure may normally be hindered from macromolecules in the solvent, perhaps by membrane phospholipids.