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.
Ménard
L
L
Howard Hughes Medical Institute Laboratories and Departments of Cell Biology, Medicine, and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Ferguson
S S
SS
Barak
L S
LS
Bertrand
L
L
Premont
R T
RT
Colapietro
A M
AM
Lefkowitz
R J
RJ
Caron
M G
MG
D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007457:Iodine Radioisotopes; D007668:Kidney; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010869:Pindolol / Q000378:metabolism; D017354:Point Mutation; D011412:Propanolamines / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D013379:Substrate Specificity; D014162:Transfection
D000319:Adrenergic beta-Antagonists; D007457:Iodine Radioisotopes; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D011412:Propanolamines; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; D010869:Pindolol; D020794:Receptor Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins; C033394:CGP 12177