Аннотация
G protein-coupled receptor kinase 2 (GRK2) is able to phosphorylate
a variety of agonist-occupied G protein-coupled receptors (GPCR)
and plays an important role in GPCR modulation. However, recent studies
suggest additional cellular functions for GRK2. Phosducin and phosducin-like
protein (PhLP) are cytosolic proteins that bind Gbetagamma subunits
and act as regulators of G-protein signaling. In this report, we
identify phosducin and PhLP as novel GRK2 substrates. The phosphorylation
of purified phosducin and PhLP by recombinant GRK2 proceeds rapidly
and stoichiometrically (0.82 +/- 0.1 and 0.83 +/- 0.09 mol of P(i)/mol
of protein, respectively). The phosphorylation reactions exhibit
apparent K(m) values in the range of 40-100 nm, strongly suggesting
that both proteins could be endogenous targets for GRK2 activity.
Our data show that the site of phosducin phosphorylation by GRK2
is different and independent from that previously reported for the
cAMP-dependent protein kinase. Analysis of GRK2 phosphorylation of
a variety of deletion mutants of phosducin and PhLP indicates that
the critical region for GRK2 phosphorylation is localized in the
C-terminal domain of both phosducin and PhLP (between residues 204
and 245 and 195 and 218, respectively). This region is important
for the interaction of these proteins with G beta gamma subunits.
Phosphorylation of phosducin by GRK2 markedly reduces its G beta
gamma binding ability, suggesting that GRK2 may modulate the activity
of the phosducin protein family by disrupting this interaction. The
identification of phosducin and PhLP as new substrates for GRK2 further
expands the cellular roles of this kinase and suggests new mechanisms
for modulating GPCR signal transduction.
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