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Members of the G Protein-Coupled Receptor Kinase Family That Phosphorylate the β2-Adrenergic Receptor Facilitate Sequestration

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Howard Hughes Medical Institute Laboratories and Departments of Cell Biology, Medicine, and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
Cite this: Biochemistry 1996, 35, 13, 4155–4160
Publication Date (Web):April 2, 1996
Copyright © 1996 American Chemical Society

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    We recently reported that a β2-adrenergic receptor (β2AR) 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 etal. (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 perse, but not the interaction with a specific GRK, is required to facilitate β2AR sequestration.

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     This work was supported by NIH Grant NS 19576 and by a Bristol Myers Squibb Unrestricted Grant Award to M.G.C. S.S.G.F. is the recipient of a fellowship from The Medical Research Council of Canada. L.S.B. is the recipient of a fellowship from the Howard Hughes Medical Institute.

     Department of Cell Biology, Duke University Medical Center.


     These authors contributed equally to this work.

     Howard Hughes Medical Institute Laboratories.

     Department of Biochemistry, Duke University Medical Center.


     To whom correspondence should be addressed at the Duke University Medical Center, Box 3287, Durham, NC 27710. Telephone:  (919) 684-5433. Fax:  (919) 681-8641.

     Abstract published in Advance ACS Abstracts, March 15, 1996.

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