Members of the G protein-coupled receptor kinase family that phosphorylate the beta2-adrenergic receptor facilitate sequestration.

We recently reported that a beta2-adrenergic receptor (beta2AR) mutant, Y326A, defective in its ability to sequester in response to agonist stimulation was a poor substrate for G protein-coupled receptor kinase (GRK)-mediated phosphorylation; however, its ability to be phosphorylated and sequestered could be restored by overexpressing GRK2 [Ferguson et al. (1995) ...
J. Biol. Chem. 270, 24782]. In the present report, we tested the ability of each of the known GRKs (GRK1-6) to phosphorylate and rescue the sequestration of the Y326A mutant in HEK-293 cells. We demonstrate that in addition to GRK2, GRK3-6 can phosphorylate the Y326A mutant and rescue its sequestration; however, GRK1 was totally ineffective in rescuing either the phosphorylation or the sequestration of the mutant receptor. We found that the agonist-dependent rescue of Y326A mutant phosphorylation by GRK2, -3, and -5 was associated with the agonist-dependent rescue of sequestration. In contrast, overexpression of GRK4 and -6 led mainly to agonist-independent phosphorylation of the Y326A mutant accompanied by increased basal receptor sequestration. Our results demonstrate that phosphorylation per se, but not the interaction with a specific GRK, is required to facilitate beta2AR sequestration.
Mesh Terms:
Adrenergic beta-Antagonists, Cell Line, Cloning, Molecular, GTP-Binding Proteins, Humans, Immunoblotting, Iodine Radioisotopes, Kidney, Phosphates, Phosphorus Radioisotopes, Phosphorylation, Pindolol, Point Mutation, Propanolamines, Receptor Protein-Tyrosine Kinases, Receptors, Adrenergic, beta-2, Recombinant Proteins, Sequence Tagged Sites, Substrate Specificity, Transfection
Biochemistry
Date: Apr. 02, 1996
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