G protein-coupled receptor kinases (GRKs) specifically phosphorylate and regulate the activated form of multiple G protein-coupled receptors. Recent studies have revealed that GRKs are also subject to regulation. In this regard, GRK2 and GRK5 can be phosphorylated and either activated or inhibited, respectively, by protein kinase C. Here we demonstrate that calmodulin, another mediator of calcium signaling, is a potent inhibitor of GRK activity with a selectivity for GRK5 (IC 50 ϳ50 nM) > GRK6 > > GRK2 (IC 50 ϳ2 M) > > GRK1. Calmodulin inhibition of GRK5 is mediated via a reduced ability of the kinase to bind to both receptor and phospholipid. Interestingly, calmodulin also activates autophosphorylation of GRK5 at sites distinct from the two major autophosphorylation sites on GRK5. Moreover, calmodulin-stimulated autophosphorylation directly inhibits GRK5 interaction with receptor even in the absence of calmodulin. Using glutathione S-transferase-GRK5 fusion proteins either to inhibit calmodulin-stimulated autophosphorylation or to bind directly to calmodulin, we determined that an amino-terminal domain of GRK5 (amino acids 20 -39) is sufficient for calmodulin binding. This domain is abundant in basic and hydrophobic residues, characteristics typical of calmodulin binding sites, and is highly conserved in GRK4, GRK5, and GRK6. These studies suggest that calmodulin may serve a general role in mediating calcium-dependent regulation of GRK activity. G protein-coupled receptor kinases (GRKs)1 form a family of serine/threonine protein kinases with the unique ability to recognize specifically the agonist-activated state of G proteincoupled receptors (1, 2). GRK-mediated phosphorylation promotes the binding of an arrestin protein, thereby uncoupling the receptor from G protein and terminating receptor signaling. Six members of the GRK family have been identified, and based on their sequence homology they have been divided into three subfamilies (2). GRK1 (rhodopsin kinase) forms one group; GRK2 (␤-adrenergic receptor kinase) and GRK3 a second; and GRK4, GRK5, and GRK6 combine into a third subfamily.All GRKs share a similar structural organization with a poorly conserved amino-terminal domain of ϳ185 residues, a conserved protein kinase catalytic domain of ϳ270 residues, and a variable length carboxyl-terminal domain of 105-230 residues (3). However, although all GRKs have a similar overall structure and function, various subfamily members also have certain unique features. For example, various GRKs utilize different mechanisms to promote membrane association, an event critical for receptor interaction. GRK1 is farnesylated (4), GRK2 and 3 interact with phospholipids and G protein ␤␥ subunits via pleckstrin homology domains (5-8), GRK4 (9) and GRK6 (10) are palmitoylated, and GRK5 binds to phospholipids via polybasic regions in the amino-and carboxyl-terminal domains (11,12).Another characteristic that appears specific for the GRK subtype involves regulation of kinase activity. For example, in the visual system, GRK1 ha...

No abstract

The G protein ␤ subunit G␤5 deviates significantly from the other four members of G␤-subunit family in amino acid sequence and subcellular localization. To detect the protein targets of G␤5 in vivo, we have isolated a native G␤5 protein complex from the retinal cytosolic fraction and identified the protein tightly associated with G␤5 as the regulator of G protein signaling (RGS) protein, RGS7. Here we show that complexes of G␤5 with RGS proteins can be formed in vitro from the recombinant proteins. The reconstituted G␤5-RGS dimers are similar to the native retinal complex in their behavior on gel-filtration and cationexchange chromatographies and can be immunoprecipitated with either anti-G␤5 or anti-RGS7 antibodies. The specific G␤5-RGS7 interaction is determined by a distinct domain in RGS that has a striking homology to G␥ subunits. Deletion of this domain prevents the RGS7-G␤5 binding, although the interaction with G␣ is retained. Substitution of the G␥-like domain of RGS7 with a portion of G␥1 changes its binding specificity from G␤5 to G␤1. The interaction of G␤5 with RGS7 blocked the binding of RGS7 to the G␣ subunit G␣o, indicating that G␤5 is a specific RGS inhibitor.Signal transduction through heterotrimeric (G␣␤␥) G proteins is governed by the cycle of GTP binding and hydrolysis by the G␣ subunit (G␣). An activated receptor catalyzes the exchange of GDP bound to G␣ initially for GTP, leading to the dissociation of G␣ from the tightly associated G␤␥-subunit complex. In this active state, the G protein modulates the activity of second messenger-generating effector enzymes and ion channels until GTP hydrolysis returns the cascade to its resting state. It has been known for a number of years that the rate of intrinsic GTPase activity of G␣ in vitro is much slower than the rate of termination of some physiological responses. Therefore, it has been proposed that additional factors accelerate GTPase activity in vivo. One class of G protein GTPaseactivating proteins (GAPs) are G protein effectors such as cGMP phosphodiesterase (1) and phospholipase C (2). Most of the G protein effector molecules, however, do not posses GAP activity. In the past 2 years, a new class of GAPs for G proteins, termed regulators of G protein signaling (RGS), has emerged (for reviews, see refs. 3-5). Thus far, about 20 RGS proteins have been discovered in mammals. RGS vary dramatically in size (from 23 to 160 kDa) and sequence, but they all have a common ''RGS domain'' (Ϸ120 aa), which is responsible for the binding to the G␣ subunits and is sufficient for the GAP activity of RGS (6, 7). The function of the other domains in the RGS proteins remains largely unexplored. However, it had been shown that RGS12 contains a PDZ domain (8), and protein p115 RhoGEF, which has a GAP activity for G␣ subunits G␣12 and G␣13, is also a guanine nucleotide-exchange factor for a small G protein, Rho (9). These findings indicate that, in addition to their interaction with G␣ subunits, RGS proteins might interact with other molecules.While inve...

Inhibition of G protein-coupled receptor kinases (GRKs) by Ca2+-binding proteins has recently emerged as a general mechanism of GRK regulation. While GRK1 (rhodopsin kinase) is inhibited by the photoreceptor-specific Ca2+-binding protein recoverin, other GRKs can be inhibited by Ca2+-calmodulin. To dissect the mechanism of this inhibition at the molecular level, we localized the GRK domains involved in Ca2+-binding protein interaction using a series of GST-GRK fusion proteins. GRK1, GRK2, and GRK5, which represent the three known GRK subclasses, were each found to possess two distinct calmodulin-binding sites. These sites were localized to the N- and C-terminal regulatory regions within domains rich in positively charged and hydrophobic residues. In contrast, the unique N-terminally localized GRK1 site for recoverin had no clearly defined structural characteristics. Interestingly, while the recoverin and calmodulin-binding sites in GRK1 do not overlap, recoverin-GRK1 interaction is inhibited by calmodulin, most likely via an allosteric mechanism. Further analysis of the individual calmodulin sites in GRK5 suggests that the C-terminal site plays the major role in GRK5-calmodulin interaction. While specific mutation within the N-terminal site had no effect on calmodulin-mediated inhibition of GRK5 activity, deletion of the C-terminal site attenuated the effect of calmodulin on GRK5, and the simultaneous mutation of both sites rendered the enzyme calmodulin-insensitive. These studies provide new insight into the mechanism of Ca2+-dependent regulation of GRKs.