Direct effect of insulin on the binding of calmodulin to rat adipocyte plasma membranes

Abstract

The interaction of calmodulin with its binding proteins on the adipocyte plasma membrane has previously been described (Goewert, R. R., Landt, M., and McDonald, J. M. (1982) Biochemistry 21, 5310-5315). In this paper we report that insulin directly affects specific calcium-dependent calmodulin binding to adipocyte plasma membranes. The direct effect of insulin on total calcium-dependent 125I-calmodulin binding was studied using 0.8 microM calmodulin. Insulin (100 microunits/ml) directly stimulated the binding of calmodulin by 19.6 +/- 2.3% (n = 6, p less than 0.001) at steady state, whereas the relatively inactive insulin analogue, desoctapeptide insulin, at equimolar concentrations had no effect. Analysis of Scatchard plots indicated that insulin increased the number of high affinity binding sites on the membrane without altering the affinity of these sites. The effect of insulin on the high affinity calmodulin binding was dependent upon increasing concentrations of insulin between 0 and 60 microunits/ml. A maximum stimulation of 75 +/- 17% (n = 4) of calcium-dependent calmodulin binding was observed at 40 microunits/ml of insulin. Effects of insulin were observed within 5 min of initiating 125I-calmodulin binding. These effects of insulin were most prominent above the K0.5 for calcium (approximately 2.0 microM). These direct effects of insulin on high affinity calmodulin binding suggest that the intracellular redistribution of calmodulin may play an important role in the early regulatory events directed by insulin on cellular metabolism.