Quantitative Aspects of the Insulin-Receptor Interaction in Liver Plasma Membranes

C Ronald Kahn,David M Neville,Pierre Freychet,Jesse Roth

doi:10.1016/s0021-9258(19)42825-1

Abstract

Abstract The interaction of insulin with a purified and well characterized preparation of liver plasma membranes has been studied. The time course of binding of 125I-insulin and displacement of tracer at the plateau of binding was measured as a function of temperature and ionic strength. Hormone tracer concentrations were in the low physiologic range. The reaction is complex and includes reversible binding of insulin to its receptor and degradation of both insulin and receptor. The degrading reactions were minimized by using low to medium membrane concentrations and were corrected for by measuring the intact insulin remaining. When the binding reaction was studied over the range of 0.06 to 1000 ng per ml of insulin at 30°, the data fit a model with a minimum of three classes of receptor sites: a high affinity low capacity site with a K of 2.0 x 109 m-1 and a capacity of 5 x 10-14 moles per mg of membrane protein; a low affinity-high capacity site with a K of 2.1 x 108 m-1 and a capacity of 1.5 x 10-12 moles per mg of membrane protein; and a very low affinity-high capacity site (nonspecific site) representing about 5% of the total tracer binding. Both the affinity and binding capacity are influenced by temperature and ionic strength. Kinetic data for insulin receptor complex dissociation are also consistent with heterogeneity of receptor sites. Discrepancies in the literature between the number of reported classes of insulin receptor sites and their affinity constants are discussed in terms of the variables of this complex reaction.

Highlights

In an attempt to resolve some of these differences and to establish a basis for the comparison of insulin-receptor interaction in different physiologic states, we have studied the quantitative aspects of insulin interaction with its receptor in a highly purified plasma membrane preparation from rat liver (21)
We have directed our attention to the methods of analysis of the data and to the complexity of the reaction. In this preparation we find a population of receptor sites which is heterogenous with respect to affinity constants
When the data are corrected by taking insulin degradation (Fig. 2B) into account, the binding curves show a react,ion which reaches its maximum at 60 min and an apparent equilibrium persists for at least 2 hours (Fig. 2C)

Summary

Methods

The methods employed in the binding studies have been previously described in detail (2). Y-insulin (specific activity 150 to 200 &i per pg) was incubated with purified plasma membranes of rat liver (21) in a Krebs-Ringer phosphate buffer, pH 7.5. Bovine serum albumin and concentrations of hormone and membrane as indicated in the legends to the figures and t)ables. The membrane-bound hormone was separated by centrifugation of duplicate or triplicate samples through 200 /*l of chilled buffer. Degradation of the unbound insulin in the supernatant was measured by loss of ability to bind to antiinsulin antibodies or to fresh aliquots of membrane (22). Purified plasma membranes and 1251-insulin were prepared as previously described (21, 22). The source of all other materials has been given (2, 21, 22)

Results

Discussion

Conclusion