Peroxovanadate (PVN) is an insulin-like agent that inhibits the dephosphorylation of the insulin receptor kinase. PVN inhibited the lipolytic action of 0.1 μM isoproterenol by 88%, which is a relatively specific β1 catecholamine agonist at this concentration, but was largely ineffective against β3 agonists or forskolin. To determine whether PVN-mediated desensitization of the β1AR was associated with enhanced phosphorylation, we immunoprecipitated the β1AR from rat adipocytes that were metabolically labeled with 32PO4. Isoproterenol enhanced the net phosphorylation of the β1AR by 8 ± 2-fold over control. PVN increased the net phosphorylation of the β1AR by 5 ± 0.5-fold, and together with isoproterenol, they enhanced the phosphorylation of the β1AR by 2-fold over isoproterenol alone. Phosphoamino acid analysis of the phosphorylated receptor revealed phosphate incorporation into serine that was proportional to the radioactivity incorporated into the immunoprecipitated receptor. PVN inhibited the serine/threonine phosphatase calcineurin, suggesting that inhibition of receptor dephosphorylation may play a role in the actions of PVN. Cyanogen bromide cleavage of the phosphorylated β1AR generated a phosphoprotein with a molecular mass consistent with carboxyl-terminal phosphorylation. Furthermore, the magnitude of receptor phosphorylation by isoproterenol was 3-fold larger than that due to forskolin, suggesting that the β1AR is a substrate for the βAR kinase that phosphorylates carboxyl-terminal residues in the β2AR. Our findings suggest that PVN may be a powerful new tool with which to study the phosphorylation of other G protein-coupled receptors.
|Original language||English (US)|
|Number of pages||9|
|State||Published - Jun 1 1996|
ASJC Scopus subject areas
- Molecular Medicine