title: 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.
abstract: The aim of the present study was to elucidate events in the plasma membrane (PM) associated with the previously described effect of insulin to rapidly enhance the number of cell surface insulin binding sites in rat adipocytes. [125I]insulin was cross-linked to cell surface insulin receptors of intact cells that had been preincubated with or without insulin. Subsequently prepared PM displayed a approximately 3-fold increase in bound [125I]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 receptor alpha-subunit. The magnitude of the effect was similar to that found for insulin binding to intact cells that had been preincubated with insulin. In contrast, the insulin binding capacity in the PM was not affected by prior treatment of cells with insulin when assessed with the addition of [125I]insulin directly to solubilized PM; this suggests an unchanged total number of PM receptors. Thus, the enhancement of 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, mimicked the effect of insulin to enhance cell surface binding in adipocytes, and this suggests a pool of cryptic PM receptors. Both the nonmetabolizable cAMP analog N6-monobutyryl cAMP (N6-mbcAMP) and the serine/threonine phosphatase inhibitor okadaic acid abolished the effect of concomitant insulin treatment to increase binding capacity. In contrast, the tyrosine phosphatase inhibitor vanadate increased insulin binding even in the presence of okadaic acid or N6-mbcAMP. The effect of N6-mbcAMP to impair cell surface insulin binding was also evident in the presence of a peptide derived from the major histocompatibility complex type I that effectively impairs receptor internalization, but the amount of PM receptors assessed by immunoblot was unaltered. Taken together, the data suggest that insulin exposure leads to the uncovering of cryptic receptors associated with the PM. It is also suggested that tyrosine phosphorylation promotes this process, whereas enhanced serine phosphorylation, e.g. produced by cAMP, impairs the functional insertion of the receptors, rendering them unable to bind insulin.
authors.affiliation: Lundberg Laboratory for Diabetes Research, Department of Medicine, Sahlgrenska University Hospital, Goteborg, Sweden.
authors.forename: J W
authors.initials: JW
authors.lastname: Eriksson
authors.forename: P
authors.initials: P
authors.lastname: Lönnroth
authors.forename: C
authors.initials: C
authors.lastname: Wesslau
authors.forename: U
authors.initials: U
authors.lastname: Smith
mesh_terms: D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D015395:Histocompatibility Antigens Class I / Q000494:pharmacology; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D008297:Male; D019319:Okadaic Acid / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011972:Receptor, Insulin / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D015395:Histocompatibility Antigens Class I; D007328:Insulin; D010750:Phosphoproteins; D010769:Phosphothreonine; D010768:Phosphoserine; D019319:Okadaic Acid; D019000:Phosphotyrosine; D000242:Cyclic AMP; D011972:Receptor, Insulin; D010738:Type C Phospholipases