G-protein-coupled receptors are desensitized by a two-step process. In a first step, G-protein-coupled receptor kinases (GRKs) phosphorylate agonist-activated receptors that subsequently bind to a second class of proteins, the arrestins. GRKs can be classified into three subfamilies, which have been implicated in various diseases. The physiological role(s) of GRKs have been difficult to study as selective inhibitors are not available. We have used SELEX (systematic evolution of ligands by exponential enrichment) to develop RNA aptamers that potently and selectively inhibit GRK2. This process has yielded an aptamer, C13, which bound to GRK2 with a high affinity and inhibited GRK2-catalyzed rhodopsin phosphorylation with an IC 50 of 4.1 nM. Phosphorylation of rhodopsin catalyzed by GRK5 was also inhibited, albeit with 20-fold lower potency (IC 50 of 79 nM). Furthermore, C13 reveals significant specificity, since almost no inhibitory activity was detectable testing it against a panel of 14 other kinases. The aptamer is two orders of magnitude more potent than the best GRK2 inhibitors described previously and shows high selectivity for the GRK family of protein kinases.

G-protein-coupled receptor kinases (GRKs) are important regulators of G-protein-coupled receptor function. Two members of this family L, GRK2 and GRK5 L, have been shown to be substrates for protein kinase C (PKC).Whereas PKC-mediated phosphorylation results in inhibition of GRK5, it increases the activity of GRK2 toward its substrates probably through increased affinity for receptor-containing membranes. We show here that this increase in activity may be caused by relieving a tonic inhibition of GRK2 by calmodulin. In vitro, GRK2 was preferentially phosphorylated by PKC isoforms ␣, ␥, and ␦. Two-dimensional peptide mapping of PKC␣-phosphorylated GRK2 showed a single site of phosphorylation, which was identified as serine 29 by HPLC-MS. A S29A mutant of GRK2 was not phosphorylated by PKC in vitro and showed no phorbol ester-stimulated phosphorylation when transfected into human embryonic kidney (HEK)293 cells. Serine 29 is located in the calmodulinbinding region of GRK2, and binding of calmodulin to GRK2 results in inhibition of kinase activity. This inhibition was almost completely abolished in vitro when GRK2 was phosphorylated by PKC. These data suggest that calmodulin may be an inhibitor of GRK2 whose effects can be abolished with PKC-mediated phosphorylation of GRK2. Signaling via G-protein-coupled receptors (GPCRs)1 is subject to a variety of regulatory processes. One of the key regulatory mechanisms is the control of receptor function by Gprotein-coupled receptor kinases (GRKs). These kinases phosphorylate the agonist-bound state of GPCRs, and this phosphorylation is often the initial step in homologous receptor desensitization, the loss of receptor responsiveness upon repeated or prolonged receptor stimulation (1, 2). To date, six members of the GRK family are known (3, 4). Of these, GRK2 (previously called ␤-adrenergic receptor kinase-1) as well as GRK3, GRK5, and GRK6 are widely expressed throughout the mammalian body whereas GRK1 (also known as rhodopsin kinase) and GRK4 have been found only in specific tissues.All GRKs have a similar molecular architecture. A central catalytic domain is flanked by an N-and C-terminal domain. The function of the latter is to provide a membrane anchor, which seems to be essential for receptor phosphorylation (5). The C terminus of GRK1 is farnesylated, whereas GRK2 and GRK3 possess a pleckstrin homology domain implicated in phosphatidylinositol bisphosphate and G-protein ␤␥-subunit binding. GRK4 and GRK6 are palmitoylated, and GRK5 binds phospholipids through a poorly defined polybasic domain (3, 4). The function of the N terminus is less clear. It has been shown for GRK1 that antibodies against an epitope in the N terminus prevent phosphorylation of light-activated rhodopsin but not of a peptide substrate (6). However, the epitope against which the antibodies were directed is poorly conserved in the GRK family. Recent results have indicated that the N terminus of GRK2 and GRK3 contains an RGS (regulator of G-protein signaling) domain that specifically binds to and act...

G-protein-coupled receptor kinase 2 (GRK2) is activated by free G␤␥ subunits. A G␤␥ binding site of GRK2is localized in the carboxyl-terminal pleckstrin homology domain. This G␤␥ binding site of GRK2 also regulates G␤␥-stimulated signaling by sequestering free G␤␥ subunits. We report here that truncation of the carboxyl-terminal G␤␥ binding site of GRK2 did not abolish the G␤␥ regulatory activity of GRK2 as determined by the inhibition of a G␤␥-stimulated increase in inositol phosphates in cells. This finding suggested the presence of a second G␤␥ binding site in GRK2. And indeed, the amino terminus of GRK2 (GRK2 1-185 ) inhibited a G␤␥-stimulated inositol phosphate signal in cells, purified GRK2 1-185 suppressed the G␤␥-stimulated phosphorylation of rhodopsin, and GRK2 1-185 bound directly to purified G␤␥ subunits. The amino-terminal G␤␥ regulatory site does not overlap with the RGS domain of GRK-2 because GRK2 1-53 with truncated RGS domain inhibited G␤␥-mediated signaling with similar potency and efficacy as did GRK2 1-185 . In addition to the G␤␥ regulatory activity, the amino-terminal G␤␥ 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 G␤␥ subunits, strongly suggesting that G␤␥ binding to the amino terminus of GRK2 enhances the kinase activity toward rhodopsin. Thus, the amino-terminal domain of GRK2 is a previously unrecognized G␤␥ binding site that regulates GRK2-mediated receptor phosphorylation and inhibits G␤␥-stimulated signaling.Activated G-protein-coupled receptors are switched off by phosphorylation through G-protein-coupled receptor kinases (GRKs) 1 (1). GRKs are modular proteins consisting of at least three structural domains with different functions. The core kinase domain of GRK2 and GRK3, which represents the ␤-adrenergic receptor kinase isozymes, is flanked by an aminoterminal domain, which contains an RGS domain, and a carboxyl-terminal domain, which contains a pleckstrin homology domain (PH domain) (2-4). The activation of GRK2 and GRK3 requires the activation and dissociation of a heterotrimeric G-protein, i.e. the kinases are activated by free G␤␥ subunits (5, 6). A G␤␥ binding site of GRK2 and GRK3 is localized in the carboxyl terminus of the kinase and overlaps the PH domain (7). Truncation of the PH domain of GRK2 generates a kinase with compromised regulation by G␤␥ subunits (7). The carboxyl-terminal G␤␥ binding site of GRK2 also regulates G␤␥-stimulated signaling by sequestering free G␤␥ subunits (8).Analyzing the G␤␥ regulatory activity of proteins is a means of identifying G␤␥-binding proteins or localizing G␤␥ binding sites of proteins (9 -11). To find out whether the G␤␥ regulatory activity of GRK2 resides entirely in the carboxyl-terminal PH domain, we analyzed the G␤␥-sequestering activity of wildtype GRK2 and of carboxyl-terminal-truncated GRK2 mutants. The capacity of thos...