The full-length, cytoplasmic C-terminus of the beta 2-adrenergic receptor expressed in E-coli acts as a substrate for phosphorylation by protein kinase A, insulin receptor tyrosine kinase, GRK2, but not protein kinase C and suppresses desensitization when expressed in vivo

The ability of the cytoplasmic, full-length C-terminus of the beta2-adrener gic receptor (BAC1) expressed in Escherichia coli to act as a functional do main and substrate for protein phosphorylation was tested. BAC1 was express ed at high-levels, purified, and examined in solution as a substrate for pr otein phosphorylation. The mobility of BAC1 on SDS-PAGE mimics that of the native receptor itself, displaying decreased mobility upon chemical reducti on of disulfide bonds. Importantly, the C-terminal, cytoplasmic domain of t he receptor expressed in E. coil was determined to be a substrate for phosp horylation by several candidate protein kinases known to regulate G-protein -linked receptors. Mapping was performed by proteolytic degradation and mat rix-assisted laser desorption ionization, time-of-flight mass spectrometry. Purified BAC1 is phosphorylated readily by protein kinase A, the phosphory lation occurring within the predicted motif RRSSSK. The kinetic properties of the phosphorylation by protein kinase A displayed cooperative character. The activated insulin receptor tyrosine kinase, which phosphorylates the b eta-adrenergic receptor in vivo phosphorylates BAC1. The Y364 residue of BA C1 was predominantly phosphorylated by the insulin receptor kinase. GRK2 ca talyzed modest phosphorylation of BAC1. Phosphorylation of the human analog of BAC1 in which Cys341 and Cys378 were mutated to minimize disulfide bond ing constraints, displayed robust phosphorylation following thermal activat ion, suggesting under standard conditions that the population of BAC1 molec ules capable of assuming the "activated" conformer required by GRKs is low. BAC1 was not a substrate for protein kinase C, suggesting that the canonic al site in the second cytoplasmic loop of the intact receptor is preferred. The functional nature of BAC1 was tested additionally by expression of BAC 1 protein in human epidermoid carcinoma A431 cells. BAC1 was found to act a s a dominant-negative, blocking agonist-induced desensitization of the beta -adrenergic receptor when expressed in mammalian cells. Thus, the C-termina l, cytoplasmic tail of this G-protein-linked receptor expressed in E. coil acts as a functional domain, displaying fidelity with regard to protein kin ase action in vivo and acting as a dominant-negative with respect to agonis t-induced desensitization. (C) 2000 Academic Press.