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VOL. 74 (4) ROLE OF G PROTEIN-COUPLED RECEPTOR ...

structure. Association with actinin, actin, calmodulin, caveolin, the Raf
kinase inhibitor protein (RKIP) or with an undetermined microsomal
component negatively modulates GRK kinase-dependent functions. In
addition, phosphorylation of GRKs at different sites and by a variety of
protein kinases has emerged as an important mechanism for regulation of
their activity, the interaction with other proteins and even protein stability
(4, 5).

REGULATION OF GRK2 BY PHOSPHORYLATION

Phosphorylation by second-messenger-modulated protein kinases (PKC
and PKA).

PKC and PKA have been shown to modulate GRK activity and
membrane targeting. “In vitro”, PKC-mediated phosphorylation of GRK2
led to an enhanced phosphorylation of receptor but not soluble peptides,
suggesting that PKC phosphorylation stimulated GRK2 translocation from
the cytosol to the plasma membrane without affecting catalytic activity. It
has been recently reported that phosphorylation of GRK2 by PKC takes
place at serine 29 (8) within the calmodulin-binding region of GRK2. It
has been suggested that in intact cells a pool of GRK2 may be tonically
inhibited by forming a complex with calmodulin, and that PKC
phosphorylation would release such inhibitory interaction and allow for
GRK2 binding to the receptor substrate (8).

On the other hand, PKA activated by Gs-coupled receptors can
directly phosphorylate GRK2, leading to an enhanced GRK2 activity
toward ß2-adrenergic receptors (9). The site of PKA phosphorylation on
GRK2 was mapped at Serine 685, which is in close proximity to the Gß?
binding domain of GRK2. In fact, PKA phosphorylation does not affect the
kinase activity, but rather enhances binding of GRK2 to Gß? subunits,
thereby facilitating membrane targeting of GRK2 and interaction with
activated receptors.

Phosphorylation by c-Src and MAPK.
c-Src can directly phosphorylate GRK2 “in vitro” with high affinity

and also promotes tyrosine phosphorylation of GRK2 upon agonist

5