Abstract
G-protein-coupled receptor kinase 2 (GRK2) is activated by free Gbetagamma
subunits. A Gbetagamma binding site of GRK2 is localized in the carboxyl-terminal
pleckstrin homology domain. This Gbetagamma binding site of GRK2
also regulates Gbetagamma-stimulated signaling by sequestering free
Gbetagamma subunits. We report here that truncation of the carboxyl-terminal
Gbetagamma binding site of GRK2 did not abolish the Gbetagamma regulatory
activity of GRK2 as determined by the inhibition of a Gbetagamma-stimulated
increase in inositol phosphates in cells. This finding suggested
the presence of a second Gbetagamma binding site in GRK2. And indeed,
the amino terminus of GRK2 (GRK2(1-185)) inhibited a Gbetagamma-stimulated
inositol phosphate signal in cells, purified GRK2(1-185) suppressed
the Gbetagamma-stimulated phosphorylation of rhodopsin, and GRK2(1-185)
bound directly to purified Gbetagamma subunits. The amino-terminal
Gbetagamma regulatory site does not overlap with the RGS domain of
GRK-2 because GRK2(1-53) with truncated RGS domain inhibited Gbetagamma-mediated
signaling with similar potency and efficacy as did GRK2(1-185). In
addition to the Gbetagamma regulatory activity, the amino-terminal
Gbetagamma binding site of GRK2 affects the kinase activity of GRK2
because antibodies specifically cross-reacting with the amino terminus
of GRK2 suppressed the GRK2-dependent phosphorylation of rhodopsin.
The antibody-mediated inhibition was released by purified Gbetagamma
subunits, strongly suggesting that Gbetagamma binding to the amino
terminus of GRK2 enhances the kinase activity toward rhodopsin. Thus,
the amino-terminal domain of GRK2 is a previously unrecognized Gbetagamma
binding site that regulates GRK2-mediated receptor phosphorylation
and inhibits Gbetagamma-stimulated signaling.
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