INSULIN PROMOTES AND CYCLIC ADENOSINE-3',5'-MONOPHOSPHATE IMPAIRS FUNCTIONAL INSERTION OF INSULIN-RECEPTORS IN THE PLASMA-MEMBRANE OF RAT ADIPOCYTES - EVIDENCE FOR OPPOSING EFFECTS OF TYROSINE AND SERINE THREONINE PHOSPHORYLATION/

The aim of the present study was to elucidate events in the plasma mem brane (PM) associated with the previously described effect of insulin to rapidly enhance the number of cell surface insulin binding sites in rat adipocytes. [I-125]insulin was cross-linked to cell surface insul in receptors of intact cells that had been preincubated with or withou t insulin. Subsequently prepared PIM displayed a similar to 3-fold inc rease in bound [I-125]insulin when cells had been pretreated with 6 nM insulin for 20 min compared to membranes from control cells, and SDS- PAGE with autoradiography showed that this occurred at the insulin rec eptor alpha-subunit. The magnitude of the effect was similar to that f ound for insulin binding to intact cells that had been preincubated wi th insulin. In contrast, the insulin binding capacity in the PM was no t affected by prior treatment of cells with insulin when assessed with the addition of [I-125]insulin directly to solubilized PM; this sugge sts an unchanged total number of PM receptors. Thus, the enhancement o f cell surface insulin binding capacity produced by insulin is not due to the translocation of receptors, but instead appears to be confined to receptors already present in the PM. The addition of phospholipase C (from Clostridium perfringens), which cleaves PM phospholipids, mim icked the effect of insulin to enhance cell surface binding in adipocy tes, and this suggests a pool of cryptic PM receptors. Both the nonmet abolizable cAMP analog N-6-monobutyryl cAMP (N-6-mbcAMP) and the serin e/threonine phosphatase inhibitor okadaic acid abolished the effect of concomitant insulin treatment to increase binding capacity. In contra st, the tyrosine phosphatase inhibitor vanadate increased insulin bind ing even in the presence of okadaic acid or N-6-mbcAMP. The effect of N-6-mbcAMP to impair cell surface insulin binding was also evident in the presence of a peptide derived from the major histocompatibility co mplex type I that effectively impairs receptor internalization, but th e amount of PM receptors assessed by immunoblot was unaltered. Taken t ogether, the data suggest that insulin exposure leads to the uncoverin g of cryptic receptors associated with the PM. It is also suggested th at tyrosine phosphorylation promotes this process, whereas enhanced se rine phosphorylation, e.g. produced by cAMP, impairs the functional in sertion of the receptors, rendering them unable to bind insulin.