Jj. Onorato et al., THE BETA-ADRENERGIC-RECEPTOR KINASE (GRK2) IS REGULATED BY PHOSPHOLIPIDS, The Journal of biological chemistry, 270(36), 1995, pp. 21346-21353
The beta-adrenergic receptor kinase (beta ARK) is a member of growing
family of G protein coupled receptor kinases (GRKs). beta ARK and othe
r members of the GRK family play a role in the mechanism of agonist-sp
ecific desensitization by virtue of their ability to phosphorylate G p
rotein-coupled receptors in an agonist-dependent manner. beta ARK acti
vation is known to occur following the interaction of the kinase with
the agonist-occupied form of the receptor substrate and heterotrimeric
G protein beta gamma subunits. Recently, lipid regulation of GRK2, GR
K3, and GRK5 have also been described. Using a mixed micelle assay, GR
K2 (beta ARK1) was found to require phospholipid in order to phosphory
late the beta(2)-adrenergic receptor. As determined with a nonreceptor
peptide substrate of beta ARK, catalytic activity of the kinase incre
ased in the presence of phospholipid without a change in the K-m for t
he peptide. Data obtained with the heterobifunctional cross-linking ag
ent N-3-[I-125] azidophenylpropionamido-S-(2-thiopyridyl)-cysteine ([I
-125]ACTP) suggests that the activation by phospholipid was associated
with a conformational change in the kinase. [I-125]ACTP incorporation
increased 2-fold in the presence of crude phosphatidylcholine, and th
is increase in [I-125]ACTP labeling is completely blocked by the addit
ion of MgATP. Furthermore, proteolytic mapping was consistent with the
modification of a distinct site when GRK2 was labeled in the presence
of phospholipid. While an acidic phospholipid specificity was demonst
rated using the mixed micelle phosphorylation assay, a notable excepti
on was observed with PIP2. In the presence of PIP2, kinase activity as
well as [(125)]ACTP labeling was inhibited. These data demonstrate th
e direct regulation of GRK2 activity by phospholipids and supports the
hypothesis that this effect is the result of a conformational change
within the kinase.