Mechanism of insulin action on membrane protein recycling: a selective decrease in the phosphorylation state of insulin-like growth factor II receptors in the cell surface membrane.

Insulin action in adipocytes leads to an increase in the steady-state number of cell surface glucose transporters and insulin-like growth factor II (IGF-II) receptors that appear to cycle continuously between the plasma membrane and a low-density membrane fraction. The IGF-II receptor could be labeled to constant specific activity by incubating adipocytes with [32P]phosphate for 2 hr. The extent of phosphorylation of IGF-II receptors in plasma membranes and in low-density microsomes was compared using 125I-labeled IGF-II binding and immunoblotting to quantitate the receptors present in each fraction. Receptors in the plasma membrane fraction of control cells incorporated approximately 1 molecule of phosphate per IGF-II binding site or 2 to 3 times more phosphate than was incorporated into IGF-II receptors in the low-density microsomes. Addition of insulin to labeled adipocytes did not change the specific activity of the gamma-phosphate of ATP but produced a specific and sharp decrease in the 32P-phosphate content of IGF-II receptors in the plasma membrane. No change due to insulin in the phosphorylation of receptors derived from low-density microsomes was observed. The insulin-mediated decrease in the [32P]phosphate content of IGF-II receptors from the plasma membrane was rapid in onset, paralleled the increase in the number of IGF-II receptors on the cell surface, and persisted for at least 30 min in the presence of insulin. Furthermore, when the effect of insulin to increase the number of IGF-II receptors in the cell surface was prevented by cooling cells to 5 degrees C, the decrease in phosphorylation of plasma membrane receptors could still be observed, indicating that this latter effect is not secondary to receptor redistribution. These data indicate that insulin inhibits one or more IGF-II receptor kinases or increases phosphatase activity, or both. Decreased phosphorylation of such insulin-sensitive plasma membrane components as IGF-II receptors may play a role in increasing their steady-state cell surface concentration, perhaps by delaying their internalization.