HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Iacovelli, L. Articles by Blasi, A. D. Search for Related Content PubMed PubMed Citation Articles by Iacovelli, L. Articles by Blasi, A. D.

Regulation of G-protein-coupled receptor kinase subtypes by calcium sensor proteins

^a Department of Molecular Pharmacology and Pathology, Consorzio Mario Negri Sud, Istituto di Ricerche Farmacologiche `Mario Negri', 66030 Santa Maria Imbaro, Italy

	ABSTRACT

TOP ABSTRACT INTRODUCTION RECEPTOR HOMOLOGOUS... REGULATION OF GRK BY... REFERENCES

 
The process of G-protein-coupled receptor (GPCR) homologous desensitization is intrinsically related to the function of a class of S/T kinases named G-protein-coupled receptor kinases (GRK). GRK family is so far composed of six cloned members, named GRK1 to 6, which are classified into three subfamilies: GRK1 is alone in the first (rhodopsin kinase subfamily), GRK2 and 3 form the second [ß-adrenergic receptor kinase (ßARK) subfamily], and GRK4, 5, and 6 constitute the third (GRK4 subfamily). Recent studies from different laboratories have demonstrated that different calcium sensor proteins (CSP) can selectively regulate the activity of GRK subtypes. In the presence of calcium, rhodopsin kinase (GRK1) is inhibited by the photoreceptor-specific CSP recoverin through direct binding. Several other recoverin homologues (including NCS 1, VILIP 1, and hippocalcin) are also able to inhibit GRK1 in a calcium-dependent manner. The ubiquitous calcium binding protein calmodulin (CaM) can inhibit GRK5 with a high affinity (IC₅₀=40–50 nM). A direct interaction between GRK5 and Ca²⁺/CaM was documented and this binding did not influence the catalytic activity of the kinase, but rather reduced GRK5 binding to the membrane. These studies suggest that CSP act as functional analogs in mediating the regulation of different GRK subtypes by Ca²⁺. This mechanism, however, is highly selective with respect to the GRK subtypes: GRK1, but not GRK2 and GRK5, is regulated by recoverin and other NCS, but GRK4, 5, and 6, which belong to the GRK4 subfamily are potently inhibited by CaM, which has little or no effect on members of other GRK subfamilies. Calcium-dependent inhibition of rhodopsin kinase by recoverin represents one of the mechanisms that control adaptation to light. For the other GPCR, CSP-GRK interaction provides a feedback mechanism that can modulate homologous desensitization of these receptors.—Iacovelli, L., Sallese, M., Mariggiò, S., De Blasi, A. Regulation of G-protein-coupled receptor kinase subtypes by calcium sensor proteins. FASEB J. 13, 1–8 (1999)

Key Words: calmodulin • calcium sensor protein • rhodopsin kinase • GRK

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RECEPTOR HOMOLOGOUS... REGULATION OF GRK BY... REFERENCES

 
ONE CELL NEEDS TO continuously communicate with other cells in order to maintain the coherent function and specialization of the system they are a part of. Cell-surface receptors perform this important task by receiving and transducing within the cell signals coming from outside. The largest known family of cell-surface receptors is that of G-protein-coupled receptors (GPCR),² which mediate the transmission of very diverse stimuli such as neurotransmitters, glycopeptides hormones, peptides, odorant molecules, and photons. The functional unit is composed of the GPCR, with the characteristic structure spanning the membrane seven times, the heterotrimeric G-protein with its - and ß-subunits (G and Gß), and the effectors that interact with G-GTP and/or Gß. In particular, the dissociated G and Gß can activate or inhibit a number of effector molecules like adenylyl cyclases, phospholipase C isoforms, ion channels, and tyrosine kinases, resulting in a variety of cellular functions (1).

The process of signal transduction mediated by GPCR must be properly regulated in order to prevent overstimulation, achieve signal termination, and render the receptor responsive to subsequent stimuli. One fundamental mechanism of regulation is receptor homologous desensitization, which occurs rapidly after receptor occupancy by the agonist. Desensitization is an adaptive mechanism of the receptors that favors the regaining of receptor responsiveness after repeated stimuli over time (2, 3).

	RECEPTOR HOMOLOGOUS DESENSITIZATION

TOP ABSTRACT INTRODUCTION RECEPTOR HOMOLOGOUS... REGULATION OF GRK BY... REFERENCES

 
The process of homologous desensitization is intrinsically related to the function of a class of serine/threonine (S/T) kinases named G-protein-coupled receptor kinases (GRK) (2–5). The term `homologous' means that the desensitization of the receptor is initiated by the activation of the same receptor by the agonist. Only the occupied receptor, unlike the unbound receptor, has the suitable conformation to be phosphorylated by GRK. Phosphorylation can occur at carboxyl terminus, as for rhodopsin and the ß₂-adrenergic receptor, or at the third intracellular loop, as for the M₂-muscarinic and ₂-adrenergic receptors (6). GRK-phosphorylated receptors are only minimally desensitized, but this phosphorylation increases the affinity for arrestin. Binding of arrestin to the receptor then induces maximal homologous desensitization. The next event is the receptor sequestration in endosomal vesicles, where it will be degraded (down-regulation) or dephosphorylated by a specific phosphatase and then recycled to the membrane (resensitization). Even though the data so far depict a convincing picture of what happens between agonist binding to the receptor and its sequestration, the biochemical details of receptor regeneration events are far less clear and are still a matter of investigation. For rhodopsin, the removal of the agonist induces the dissociation of arrestin and dephosphorylation by protein phosphatase 2A. It has been shown for other GPCR that the lower pH in the endosomal compartment is probably responsible for the phosphatase activation and receptor conformational change that promote arrestin disassembling (4). Homologous desensitization is distinct from the other process of loss of receptor responsiveness known as heterologous desensitization, which is determined mainly by second messenger-dependent kinases protein kinase A (PKA) and PKC (3). The main difference between the two processes is that activated PKA or PKC phosphorylate not only agonist-stimulated receptors, but also a number of other receptors.

Structure and function of GRK
The GRK family is composed so far of six cloned members, named GRK1 to 6 ( Fig. 1). The overall structure of GRK consists of a central core, the catalytic domain, flanked by the amino-terminal region and the carboxyl-terminal region. Based on sequence homology, these six GRK subtypes are classified into three subfamilies ( Fig. 1): GRK1 is alone in the first [rhodopsin kinase (RK) subfamily], GRK2 and 3 form the second [ß-adrenergic receptor kinase (ßARK] subfamily), whereas GRK4, 5, and 6 constitute the third (GRK4 subfamily). The catalytic domain, also containing the ATP binding site, is the most conserved region among all GRK subtypes. Sites of alternative splicing have been identified for some subtypes of GRK and this topic is still under investigation. For human GRK2 and 3, no splice variants have been discovered, and one was found in GRK1 sequence. Human GRK4 has been demonstrated to have two sites of alternative splicing—one at the amino-terminal domain (exon 2), the other at carboxyl-terminal domain (exon 15)—resulting in four splice variants (7, 8). As GRK4, 5, and 6 are closely related, alternative splicings could be predicted in the latter two GRK. So far, Elalouf et al. (9) have demonstrated the existence of splice variants in rat GRK6, which are different from the GRK4 splice variants.

View larger version (11K): [in this window] [in a new window]   Figure 1. G-protein-coupled receptor family and subfamilies. Different domains are shown: Gß binding domain in GRK2 and GRK3 (ß); CaM binding domain (CaM) in GRK4, 5, and 6. Dotted CaM in GRK4 and GRK6 means that this domain is defined by homology with GRK5. The positively charged domain at the carboxyl terminus of GRK5 (+++), presumably involved in membrane targeting, and the lipid modification of GRK1, 4 and 6 are indicated. Different splicings of GRK4 are shown: exon 2 (amino acids 18–49) and 15 (amino acids 515–562) appear in boldface.

The membrane targeting domains of GRK sub-types ( Fig. 1) are located mainly at the carboxyl terminus, but the anchoring mechanisms are somewhat different. GRK1 possesses a farnesylation site, whereas a palmitoylation site is present in both GRK4 and GRK6. GRK2 and 3 have an extended carboxyl terminus in which a PH domain is located, and they are targeted to membrane by binding to Gß. The Gß binding domain partially overlaps with the PH domain. GRK5 likely binds to the membrane phospholipids by a positively charged amino acids cluster located at the carboxyl terminus.

Mechanisms that regulate GRK activity
Phosphorylation-dependent homologous desensitization is a general mechanism that regulates GPCR-mediated signaling. Given the extraordinarily large number of GPCR identified so far (1000) and the relatively small number of GRK (six genes), it appears that the interaction between these proteins is not based on a `one kinase for one receptor' rule. Most cell types have a variety of GPCR on the surface and express more than one GRK that could phosphorylate and regulate these receptors. Different GPCR can be phosphorylated by the same GRK subtype, indicating that receptor specificity is not the key determinant for kinase–receptor interaction. Therefore, it is possible to hypothesize the existence of intracellular mechanisms able to regulate the activity of GRK and to provide some degree of selectivity in the interaction between GPCR and receptor kinases ( Fig. 2).

View larger version (63K): [in this window] [in a new window]   Figure 2. Regulated interaction between G-protein-coupled receptors and GRK.

Several mechanisms able to modulate the activity of GRK have been identified. As an initial step, the stimulated receptor (i.e., agonist-occupied) is able to activate GRK (10, 11). Conformational changes of the receptors induced by agonist binding cause the exposure of two physically and functionally distinct domains. One contains the sequence that is phosphorylated by GRK and the second works as an activator of these kinases. For example in the M₂-muscarinic receptor, the sites phosphorylated by GRK2 and the domains able to activate this kinase were found to be located in different intracellular regions of the receptor (11).

PIP₂ and Gß, which are major determinants for GRK membrane targeting, are also potent activators of these kinases (12–14). PIP₂ enhances the ability of GRK subtypes to phosphorylate receptor substrate. It directly interacts with GRK and facilitates membrane association of the kinase. Although the structure and location of the PIP₂ binding site are distinct in GRK subfamilies, the activation by PIP₂ was documented with virtually all the GRK subtypes (GRK1 was not tested). In contrast, the ability of Gß to enhance kinase activity was seen exclusively with the ßARK subfamily members GRK2 and GRK3. Only GRK2 and GRK3 have an extended carboxyl terminus that contains the Gß binding site (15). All other GRK have a shorter carboxyl terminus lacking either the PH domain or the Gß binding sequence.

Intracellular calcium can modulate GRK activity by different mechanisms. For example, a rise in cytosolic calcium activates PKC, which can phosphorylate different GRK (16–18). Phosphorylation by PKC has opposing functional effects on GRK subtypes: GRK2 kinase activity is increased whereas GRK5 is substantially inhibited.

Increased intracellular calcium also binds to and activates intracellular proteins known as calcium sensor proteins (CSP). Different CSP can selectively regulate the activity of GRK subtypes, and this article will review recent findings on this topic.

	REGULATION OF GRK BY CALCIUM SENSOR PROTEINS

TOP ABSTRACT INTRODUCTION RECEPTOR HOMOLOGOUS... REGULATION OF GRK BY... REFERENCES

 
Calcium sensor proteins
Interaction of Ca²⁺ with a large number of Ca²⁺ binding proteins represents one of the mechanisms by which this second messenger controls many biological processes (19, 20). Calcium binds with high affinity to these Ca²⁺ binding proteins and induces conformational changes that enable these proteins to interact and regulate a variety of targets. One class of these proteins shares a common Ca²⁺ binding motif, the EF-hand (20). This structural motif, first identified in the crystal structure of parvalbumin, consists of two perpendicularly placed -helices (helices E and F in parvalbumin) and one interhelical loop, which together form a single Ca²⁺ binding site. The mechanism of this molecular switch lies in the conformational change induced by Ca²⁺ binding. EF-hand proteins with regulatory roles are often termed CSP, whereas those involved in Ca²⁺ buffering and transport are termed Ca²⁺ buffer proteins (see ref 20 for review). CSP represent a heterogeneous class of proteins that includes calmodulin (CaM), neuron-specific calcium sensor proteins called neuronal calcium sensors (NCS) such as recoverin, visinlike protein (VILIP), neurocalcin, hippocalcin, and the recently identified S100 family members.

Interaction between recoverin and RK (GRK1)
The first evidence of interaction and regulation of GRK by a CSP was obtained in studies of retinal phototransduction. Confirming earlier observations by Kawamura (21), several studies have suggested that the Ca²⁺ effect on cGMP phosphodiesterase is likely mediated by recoverin, which was able to decrease light-dependent rhodopsin phosphorylation (22, 23). This resulted in a prolonged lifetime of catalytically active rhodopsin and, therefore, in a larger number of transducin molecules activated by rhodopsin. Recoverin (also called S-modulin in the frog) is a myristoylated CSP expressed predominantly in vertebrate photoreceptor cells.

The molecular mechanisms of recoverin inhibition of rhodopsin phosphorylation was subsequently elucidated (24, 25). It was found that recoverin acts directly on RK to decrease its catalytic activity and that no components other than rhodopsin, RK, and recoverin are required. Inhibition of rhodopsin phosphorylation by recoverin was Ca²⁺ dependent. The covalently attached myristoyl residue enhanced the inhibitory effect of recoverin, as determined in an assay of phosphorylation of urea-stripped rod outer segments (ROS) membranes by purified recombinant RK. The IC₅₀ for myristoylated recoverin was 0.8 µM and the IC₅₀ for nonacylated recoverin was 8 µM at saturating Ca²⁺ concentrations.

Since myristoylated recoverin was a better inhibitor than nonacylated recoverin, it was suggested that the interaction of recoverin with ROS membranes enhances the inhibitory effect. However, this point is still controversial and requires further investigation (24, 26). In another study it was found that the inhibitory effect of recoverin was not affected by changing the concentration of bleached rhodopsin or the amount of membranes, leading Klenchin et al. (24) to conclude that recoverin action does not require binding to rhodopsin or ROS membranes.

Physical interaction between recoverin and RK likely represents the mechanism of the inhibition of the kinase. Recoverin binds directly to RK in a Ca²⁺-dependent manner. This is a high-affinity interaction, and immobilized recoverin was used as affinity matrix to purify RK (25).

Interaction between CaM and ßARK family members (GRK2 and 3)
While investigating whether the interaction and regulation of GRK by CSP is a general mechanism, three different laboratories have found that GRK2 and GRK3 are inhibited by CaM in a calcium-dependent manner (27–29). The IC₅₀ was 2 µM. CaM is an acidic protein which is considered the primary `decoder' of Ca²⁺ information in the cell (30, 31), exerting many of its functions when bound to Ca²⁺ (four Ca²⁺ ions per CaM molecule). Numerous proteins have been identified to be regulated by Ca²⁺/CaM, e.g., kinases, phosphodiesterases, calcium pumps, and adenylyl cyclase. Gß is also a binding target of Ca²⁺/CaM whereas the binding of GRK2 and 3 to Gß is critical in mediating the activation of these two GRK subtypes. Therefore, Gß was proposed as a possible site of indirect interaction between GRK2 and 3 with Ca²⁺/CaM. However, CaM was able to inhibit GRK2 kinase activity even in the absence of Gß, indicating that the effect of CaM is not due to sequestration of Gß from GRK2 (28, 29). Rather, preliminary data indicate that the inhibition is the result of a direct interaction between CaM and GRK2.

Interaction between CaM and GRK4 subfamily members (GRK4, 5, and 6)
The analysis of different GRK isoforms led to the unexpected observation that CaM is a potent inhibitor of GRK5 (27, 29). The IC₅₀ was 40–50 nM, indicating that CaM is ~50-fold more potent in inhibiting GRK5 kinase activity than GRK2 and 3. The other two members of this GRK subfamily, GRK4 and GRK6, were also strongly inhibited by CaM. The inhibitory effect of CaM on GRK4 (8) and GRK6 (29) was ~3-fold lower than that on GRK5 and ~30-fold more potent than that on GRK2. For GRK4 the calculated IC₅₀ was 80 nM (8).

The high-affinity interaction between CaM and GRK5 was extensively characterized. CaM inhibited the ability of GRK5 to phosphorylate bovine retinal ROS in a calcium-dependent manner, and this effect was prevented by the CaM inhibitor peptide CaMBd. A direct interaction between GRK5 and Ca²⁺/CaM was demonstrated using CaM-conjugated Sepharose 4B and confirmed using surface plasmon resonance (SPR) technology on a BIAcore instrument. The calculated K_d for this interaction was ~10 nM, a value in good agreement with the IC₅₀ for CaM-dependent inhibition of ROS phosphorylation. This binding does not influence the catalytic activity of GRK5 as demonstrated by the lack of inhibitory effect on its phosphorylating activity on the soluble GRK substrate casein. Instead, Ca²⁺/CaM significantly reduced kinase binding to the membrane and to phospholipid vesicles. Kunapuli et al. (32) have demonstrated that binding of GRK5 to phospholipids is critical in activating the kinase. Thus, inhibition of GRK5 association with the membrane by Ca²⁺/CaM would hinder with kinase activation, hence the observed inhibitory effect on receptor phosphorylation.

Direct binding of CaM to GRK4 was also documented (8). However, the inhibitory effect of CaM on membrane binding was more evident for GRK5 than for GRK4 (M. Sallese and A. De Blasi, unpublished observations) and likely accounts for the ~threefold more potent inhibitory effect of CaM on rhodopsin phosphorylation by GRK5 than by GRK4 and by GRK6. Unlike GRK4 and GRK6, which utilize covalent lipid modification to enhance binding to phospholipid membranes, GRK5 appears to interact directly with phospholipids via regions rich in basic amino acids (4, 5). This different mechanism of membrane targeting provides a likely explanation for different sensitivity to CaM. However, additional mechanisms are probably important for the inhibitory effects of CaM on GRK (29). For example it was observed that the IC₅₀ of CaM to inhibit GRK5 binding to ROS membranes (~300–400 nM) was some six- to eightfold higher that the IC₅₀ for rhodopsin phosphorylation. Moreover, at high CaM concentrations (2 µM), ~20% of the kinase remained bound to ROS membranes even though rhodopsin phosphorylation was reduced by >99%. Also CaM inhibited GRK5 phosphorylation of the soluble substrate phosvitin (IC₅₀ ~600 nM), suggesting that CaM interacts with regions of GRK5 that are likely involved in substrate binding. Together, these results indicate that CaM can directly compete for both the lipid and receptor binding sites of GRK5.

Unexpectedly it was found that CaM activates GRK5 autophosphorylation (29). It was already known that phospholipids can activate GRK5 autophosphorylation and that this resulted in activation of this kinase. However, GRK5 autophosphorylation activated by CaM was found to occur on S and T residues different from the residue (S⁴⁸⁴ and T⁴⁸⁵) substrate for phospholipid-induced autophosphorylation. Indeed, CaM-activated GRK5 autophosphorylation was observed even after mutation of S⁴⁸⁴ an T⁴⁸⁵ into aspartic acid. It was found that autophosphorylation is not the major determinant in inhibiting GRK5 activity, but it may enhance the sensitivity of GRK5 to CaM (29).

The following model of GRK5 regulation by CaM was proposed (29). At resting Ca²⁺ concentrations, GRK5 is active and able to phosphorylate agonist-occupied receptors. When a cell is stimulated and intracellular Ca²⁺ levels rise, CaM binds to GRK5 and directly inhibits receptor phosphorylation. However, since CaM-stimulated autophosphorylation also inhibits GRK5 activity, the kinase should remain inhibited even when Ca²⁺ levels go down and CaM dissociates from the enzyme. Presumably, GRK5 will eventually be dephosphorylated and return to its basal level of activity. Thus, CaM-stimulated autophosphorylation may prolong the inhibitory effect of a transient increase of intracellular Ca²⁺ levels on GRK5. A similar regulatory cycle has been demonstrated for CaM-kinase II (33).

The CaM binding site in GRK5 was identified (29) and found to be located near the amino terminus of the kinase (amino acids 20–39). GRK5-GST fusion proteins lacking this domain were unable to inhibit CaM-induced GRK5 autophosphorylation. This CaM binding site of GRK5 was confirmed by using SPR technology on a BIAcore instrument. Most of the CaM binding sites identified contain a basic amphipathic -helical structure with a large number of positively charged residues as well as hydrophobic residues that repeat with a three to four period (30, 31). Consistent with this model, helical wheel projection of the region of GRK5 that binds CaM shows the segregation of basic and hydrophobic residues to opposite sides of the helix, thereby making them available for interaction with acidic and hydrophobic patches of CaM.

The sequence corresponding to the CaM binding site was conserved within the GRK4 subfamily (GRK4, 5, and 6), which all interact with CaM at high affinity. By contrast, the putative binding site is poorly conserved in GRK1, 2, and 3, which interact with CaM at much lower affinity. Residues 22–39 of GRK5 were suggested to be involved in binding to PIP₂, which leads to the activation of these kinases (14). Since the putative PIP₂ and CaM binding domains are substantially overlapping, CaM could compete for binding of PIP₂ to GRK5. According to this possibility, the experimental evidences show that CaM inhibits direct binding of the kinase to phospholipid vesicles.

In GRK4, the region homologous to the CaM binding site of GRK5 lies entirely within exon 2, which is alternatively spliced (8). GRK4ß and GRK4 subtypes, which do not contain this sequence, were unable to bind to CaM, further supporting the role of this sequence as a CaM interacting site. However, additional sites are likely important for CaM–GRK4 physical interaction. This was indicated by the finding that GRK4, which contains the sequence encoded by exon 2 but lacks exon 15 (located near the carboxyl terminus), also was unable to bind to CaM (8).

Selectivity in the regulation of GRK by CSP
These studies suggest that CSP act as functional analogs in mediating the regulation of different GRK subtypes by Ca²⁺. Preliminary studies also indicate that S100 protein is able to inhibit GRK2 in a calcium-dependent manner (34), suggesting that several classes of EF-hand CSP can regulate GRK activity. This mechanism is, however, highly selective with respect to the different CSP and GRK subtypes (see Table 1).

In parallel experiments, Chen et al. (25) first showed that recoverin, which inhibited GRK1 phosphorylating activity by direct binding to this kinase, did not interact with GRK2 and was ineffective on ROS phosphorylation by this kinase subtype. Recoverin is a member of the NCS family; several other members, including NCS 1, VILIP 1, and hippocalcin, are also able to inhibit GRK1 in a calcium-dependent manner (26). However, these NCS are not able to inhibit GRK5 kinase activity ( Fig. 3). An analysis of VILIP in olfactory neurons indicated that, in these cells, VILIP does not interfere with GRK3 (35). Since GRK2 is the relevant GRK subtype in olfactory neurons, these results further confirm that VILIP selectively inhibits GRK1 (26) and does not affect either GRK3 (35) or GRK5 ( Fig. 3). Taken together, these data indicate that recoverin and other NCS can selectively inhibit GRK1 and do not affect the other GRK subtypes. By contrast, CaM strongly inhibits GRK4 subfamily members whereas it has little (for GRK2 and 3) or no (for GRK1) effect on the other GRK. This profile of selectivity is consistent with the tissue distribution of these proteins. Recoverin, which is a retinal protein, preferentially regulates rhodopsin kinase (GRK1) whereas the ubiquitously expressed CaM preferentially interacts with the GRK5 found in a large variety of tissues and cells.

View larger version (31K): [in this window] [in a new window]   Figure 3. GRK5 phosphorylating activity is selectively inhibited by CaM. Urea-treated ROS was incubated with 10 nM pure GRK5 under phosphorylating conditions, and phosphorylated rhodopsin (Opsin) was revealed by autoradiography after polyacrylamide gel electrophoresis. The effect of VILIP, neuronal calcium sensor 1 (NCS1), CaM, recoverin (RECOV), and hippocalcin (HIP) was evaluated in the presence or absence of Ca2+ (1 mM). CaM concentration was 0.5 µM whereas the concentration of all the other calcium binding proteins was 2 µM.

Functional implications
The functional consequences of recoverin–RK interaction were intensively investigated. Calcium ions regulate several steps of the phototransduction process by modifying the activity of different Ca²⁺ binding proteins, which in turn interact with key enzymes in the cascades (36, 37). Several pathways are activated when the concentration of Ca²⁺ decreases after photon absorption, and they all lead to negative regulation of the effect of light. These feedback mechanisms speed up the recovery of the response to light and diminish the effect of illumination, thus enabling the cell to adapt to background light. Recoverin is involved in one such mechanism. Reduction of Ca²⁺ levels by photon absorption uncouples recoverin from RK, thus allowing the inhibitory effect of rhodopsin phosphorylation to proceed. This results in a shorter lifetime of rhodopsin in photolyzed state, which is one mechanism of light adaptation. Some observations, however, suggest that the above model might represent an oversimplified and perhaps incorrect view (36). For example, in transgenic mice the recoverin knockout had no major effects on the response of the cell to light flashes. But these data must be interpreted with caution, since the alteration of the recoverin gene could result in an unexpected compensation by other components of the phototransduction cascade. In recent studies, Senin et al. (38, 39) found that recoverin inhibits the phosphorylation of dark-adapted rhodopsin better than that of bleached rhodopsin. It was proposed that the role of recoverin-dependent regulation of RK is to prevent the enzyme from participating in the unwanted phosphorylation of dark-adapted rhodopsin (this phenomenon is known as `high-gain' phosphorylation) (38, 39).

The functional consequences of regulation of GRK subtypes by CaM can be hypothesized. A number of GPCR are substrates of GRK2, 3, and 5; some of these receptors are coupled to fluctuations in intracellular Ca²⁺ concentrations, e.g., substance P, angiotensin II receptors. CaM can therefore provide a feedback mechanism for modifying homologous desensitization of these receptors. Furthermore, in any one cell, a number of receptor substrates for GRK2, 3 and 5 may be present and calcium sensors may therefore mediate a novel route of crosstalk in which one Ca²⁺ regulating receptor agonist can acutely regulate the phosphorylating activity of these GRK subtypes, thus modulating the functional efficiency of a different receptor. Only few functional data are available. Haga et al. (28) found that in transfected CHO cells the sequestration of the M₂-muscarinic receptor is attenuated by treatment with Ca²⁺ ionophore A23187. Since M₂-muscarinic receptor sequestration is a specific and sensitive sensor of GRK2 activity (40), these results indicate that the activity of GRK2 in intact cells is suppressed by increased levels of Ca²⁺ concentration.

FUTURE DIRECTIONS
The high level of redundancy of the mechanisms regulating receptor-induced intracellular responses clearly indicates that signal transduction must be highly regulated. A challenging point is to define how these different effectors work in integrated systems. Studies using intact cells or complex experimental models should be performed to define the relative role and the integrated functioning of regulatory mechanisms.

The role of intracellular calcium in modulating the regulatory proteins is becoming more and more relevant. Future studies will shed new light on further implications. For example, many other CSP could be involved in the regulation of GPCR-mediated signaling. A new class of phosphatases, named G-protein-coupled receptor phosphatases (GRP), has been recently identified. These GRP dephosphorylate desensitized receptors, thus favoring resensitization (41–43). It is interesting that these GRP possess two EF-hands and their activity is calcium dependent.

All these observations support the idea of a complex integrated network working for an efficient signaling. The identification of these players and the definition of their relative function represent a major goal in the field.

	ACKNOWLEDGMENTS

 
We are indebted to Patrick Nef (University of Geneva) for kindly providing purified CSP. This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), by C.N.R. Target Project on Biotechnology, and by EC Biomed 2 program-PL 963566. S.M. is recipient of a fellowship granted by Progetto Speciale Ricerca Scientifica e Applicata nel Mezzogiorno PS35–93/IND.

	FOOTNOTES

 
¹ Correspondence: Consorzio Mario Negri Sud, via Nazionale, 66030 S. Maria Imbaro, Italy. E-mail: DEBLASI{at}CMNS.MNEGRI.IT

² Abbreviations: ßARK, ß-adrenergic receptor kinase; CaM, calmodulin; CSP, calcium sensor proteins; GRK, G-protein-coupled receptor kinases; GPCR, G-protein-coupled receptor(s); GRP, G-protein-coupled receptor phosphatases; NCS, neuronal calcium sensors; PIP₂, phosphatidylinositol 4,5-bisphosphate; PKA, protein kinase A; RK, rhodopsin kinase; ROS, rod outer segments; SPR, surface plasmon resonance; VILIP, visinlike protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION RECEPTOR HOMOLOGOUS... REGULATION OF GRK BY... REFERENCES

 

1. Hamm, H. E. (1998) The many faces of G protein signaling. J. Biol. Chem. 273, 669–672[Free Full Text]
2. Freedman, N. J., and Lefkowitz, R. J. (1996) Desensitization of G protein-coupled receptors. Recent Prog. Horm. Res. 51, 319–353
3. Chuang, T. T., Iacovelli, L., Sallese, M., and De Blasi, A. (1996) G protein-coupled receptors: heterologous regulation of homologous desensitization and its implications. Trends Pharmacol. Sci. 17, 416–421[Medline]
4. Palczewski, K. (1997) GTP-binding-protein-coupled receptor kinases. Eur. J. Biochem. 248, 261–269[Medline]
5. Krupnick, J. G., and Benovic, J. L. (1998) The role of receptor kinases and arrestins in G protein-coupled receptor regulation. Annu. Rev. Pharmacol. Toxicol. 38, 289–319[Medline]
6. Eason, M. G., Moreira, S. P., and Liggett, S. B. (1995) Four consecutive serines in the third intracellular loop are the sites for ß-adrenergic receptor kinase-mediated phosphorylation and desensitization of the _2A-adrenergic receptor. J. Biol. Chem. 270, 4681–4688[Abstract/Free Full Text]
7. Premont, R. T., Macrae, A. D., Stoffel, R. H., Chung, N., Pitcher, J. A., Ambrose, C., Inglese, J., MacDonald, M. E., and Lefkowitz, R. J. (1996) Characterization of the G protein-coupled receptor kinase GRK4. Identification of four splice variants. J. Biol. Chem. 271, 6403–6410[Abstract/Free Full Text]
8. Sallese, M., Mariggiò, S., Collodel, G., Moretti, E., Piomboni, P., Baccetti, B., and De Blasi, A. (1997) G protein-coupled receptor kinase GRK4. Molecular analysis of the four isoforms and ultrastructural localization in spermatozoa and germinal cells. J. Biol. Chem. 272, 10188–10195[Abstract/Free Full Text]
9. Firsov, D., and Elalouf, J. M. (1997) Molecular cloning of two rat GRK6 splice variants. Am. J. Physiol. 273, C953–C961[Abstract/Free Full Text]
10. Chen, C. Y., Dion, S. B., Kim, C. M., and Benovic, J. L. (1993) ß-Adrenergic receptor kinase. Agonist-dependent receptor binding promotes kinase activation. J. Biol. Chem. 268, 7825–7831[Abstract/Free Full Text]
11. Haga, K., Kameyama, K., and Haga, T. (1994) Synergistic activation of G protein-coupled receptor kinase by G Protein ß subunits and mastroparan or related peptides. J. Biol. Chem. 269, 12594–12599[Abstract/Free Full Text]
12. Pitcher, J. A., Touhara, K., Payne, E. S., and Lefkowitz, R. J. (1995) Pleckstrin homology domain-mediated membrane association and activation of the ß-adrenergic receptor kinase requires coordinate interaction with G_ß subunits and lipid. J. Biol. Chem. 270, 11707–11710[Abstract/Free Full Text]
13. DebBurman, S. K., Ptasienski, J., Benovic, J. L., and Hosey, M. M. (1996) G protein-coupled receptor kinase GRK2 is a phospholipid-dependent enzyme that can be conditionally activated by G protein ß subunits. J. Biol. Chem. 271, 22552–22562[Abstract/Free Full Text]
14. Pitcher, J. A., Fredericks, Z. L., Stone, W. C., Premont, R. T., Stoffel, R. H., Koch, W. J., and Lefkowitz, R. J. (1996) Phosphatidylinositol 4,5-bisphosphate (PIP₂)-enhanced G protein-coupled receptor kinase (GRK) activity. Location, structure, and regulation of the PIP₂ binding site distinguishes the GRK subfamilies. J. Biol. Chem. 271, 24907–24913[Abstract/Free Full Text]
15. Koch, W. J., Inglese, J., Stone, W. C., and Lefkowitz, R. J. (1993) The binding site for the ß subunits of heterotrimeric G proteins on the ß-adrenergic receptor kinase. J. Biol. Chem. 268, 8256–8260[Abstract/Free Full Text]
16. Chuang, T. T., LeVineIII, H., and De Blasi, A. (1995) Phosphorylation and activation of ß-adrenergic receptor kinase by protein kinase C. J. Biol. Chem. 270, 18660–18665[Abstract/Free Full Text]
17. Winstel, R., Freund, S., Krasel, C., Hoppe, E., and Lohse, M. J. (1996) Protein kinase cross-talk: membrane targeting of the ß-adrenergic receptor kinase by protein kinase C. Proc. Natl. Acad. Sci. USA 93, 2105–2109[Abstract/Free Full Text]
18. Pronin, A. N., and Benovic, J. L. (1997) Regulation of the G protein-coupled receptor kinase GRK5 by protein kinase C. J. Biol. Chem. 272, 3806–3812[Abstract/Free Full Text]
19. Schafer, B. W., and Heizmann, C. W. (1996) The S100 family of EF-hand calcium-binding proteins: functions and pathology. Trends Biochem. Sci. 21, 134–140[Medline]
20. Ikura, M. (1996) Calcium binding and conformational response in EF-hand proteins. Trends Biochem. Sci. 21, 14–17[Medline]
21. Kawamura, S. (1993) Rhodopsin phosphorylation as a mechanism of cyclic GMP phosphodiesterase regulation by S-modulin. Nature (London) 362, 855–857[Medline]
22. Nikonov, S. S., Filatov, G. N., and Fesenko, E. E. (1993) On the activation of phosphodiesterase by a 26 kDa protein. FEBS Lett. 316, 34–36[Medline]
23. Gorodovikova, E. N., Senin, I. I., and Philippov, P. P. (1994) Calcium-sensitive control of rhodopsin phosphorylation in the reconstituted system consisting of photoreceptor membranes, rhodopsin kinase and recoverin. FEBS Lett. 353, 171–172[Medline]
24. Klenchin, V. A., Calvert, P. D., and Bownds, M. D. (1995) Inhibition of rhodopsin kinase by recoverin. J. Biol. Chem. 270, 16147–16152[Abstract/Free Full Text]
25. Chen, C. K., Inglese, J., Lefkowitz, R. J., and Hurley, J. B. (1995) Ca²⁺-dependent interaction of recoverin with rhodopsin kinase. J. Biol. Chem. 270, 18060–18066[Abstract/Free Full Text]
26. De Castro, E., Nef, S., Fiumelli, H., Lenz, S. E., Kawamura, S., and Nef, P. (1995) Regulation of rhodopsin phosphorylation by a family of neuronal calcium sensors. Biochem. Biophys. Res. Commun. 216, 133–140[Medline]
27. Chuang, T. T., Paolucci, L., and De Blasi A. (1996) Inhibition of G protein-coupled receptor kinase subtypes by Ca²⁺/calmodulin. J. Biol. Chem. 271, 28691–28696[Abstract/Free Full Text]
28. Haga, K., Tsuga, H., and Haga, T. (1997) Ca²⁺ dependent inhibition of G protein-coupled receptor kinase 2 by calmodulin. Biochemistry 36, 1315–1321[Medline]
29. Pronin, A. N., Satpaev, D. K., Slepak, V. Z., and Benovic, J. L. (1997) Regulation of G Protein-coupled receptor kinases by calmodulin and localization of the calmodulin binding domain. J. Biol. Chem. 272, 18273–18280[Abstract/Free Full Text]
30. James, P., Vorherr, T., and Carafoli, E. (1995) Calmodulin-binding domains: just two faced or multi-faceted? Trends Biochem. Sci. 20, 38–44[Medline]
31. Rhoads, A. R., and Friedberg, F. (1997) Sequence motifs for calmodulin recognition. FASEB J. 11, 331–340[Abstract]
32. Kunapuli, P., Gurevich, V. V., and Benovic, J. L. (1994) Phospholipid-stimulated autophosphorylation activates the G protein-coupled receptor kinase GRK5. J. Biol. Chem. 269, 10209–10212[Abstract/Free Full Text]
33. Gnegy, M. E. (1993) Calmodulin in neurotransmitter and hormone action. Annu. Rev. Pharmacol. Toxicol. 33, 45–70[Medline]
34. Haga, K., and Haga, T. (1997) Inhibition by calmodulin or S100 protein of agonist-dependent phosphorylation of m2 muscarinic receptors by G protein coupled receptor kinase 2. Life Sci. 96, 1184
35. Boekhoff, I., Braunewell, K. H., Andreini, I., Breer, H., and Gundelfinger, E. (1997) The calcium-binding protein VILIP in olfactory neurons: regulation of second messenger signaling. Eur. J. Biochem. 72, 151–158
36. Polans, A., Baehr, W., and Palczewski, K. (1996) Turned on by Ca²⁺! The physiology and pathology of Ca²⁺-binding proteins in the retina. Trends Neurosci. 19, 547–554[Medline]
37. Koutalos, Y., and Yau, K. W. (1996) Regulation of sensitivity in vertebrate rod photoreceptors by calcium. Trends Neurosci. 19, 73–81[Medline]
38. Senin, I. I., Dean, K. R., Zargarov, A. A., Akhtar, M., and Philippov, P. P. (1997) Recoverin inhibits the phosphorylation of dark-adapted rhodopsin more than it does that of bleached rhodopsin: a possible mechanism through which rhodopsin kinase is prevented from participation in a side reaction. Biochem. J. 321, 551–555
39. Senin, I. I., Zargarov, A. A., Akhtar, M., and Philippov, P. P. (1997) Rhodopsin phosphorylation in bovine rod outer segments is more sensitive to the inhibitory action of recoverin at the low rhodopsin bleaching than it is at the high bleaching. FEBS Lett. 408, 251–254[Medline]
40. Tsuga, H., Kameyama, K., Haga, T., Kurose, H., and Nagao, T. (1994) Sequestration of muscarinic acetylcholine receptor m2 subtypes. Facilitation by G protein-coupled receptor kinase (GRK2) and attenuation by a dominant-negative mutant of GRK2. J. Biol. Chem. 269, 32522–32527[Abstract/Free Full Text]
41. Pitcher, J. A., Payne, E. S., Csortos, C., DePaoli-Roach, A. A., and Lefkowitz, R. J. (1995) The G-protein-coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity. Proc. Natl. Acad. Sci. USA 92, 8343–8347[Abstract/Free Full Text]
42. Vinós, J., Jalink, K., Hardy, R. W., Britt, S. G., and Zuker, C. S. (1997) A G protein-coupled receptor phosphatase required for rhodopsin function. Science 277, 687–690[Abstract/Free Full Text]
43. Montini, E., Rugarli, E. I., Van de Vosse, E., Andolfi, G., Mariani, M., Puca, A. A., Consalez, G. G., den Dunnen, J. T., Ballabio, A., and Franco, B. (1997) A novel human serine-threonine phosphatase related to the Drosophila retinal degeneration C (rdg C) gene is selectively expressed in sensory neurons of neural crest origin. Hum. Mol. Gen. 6, 1137–1145[Abstract/Free Full Text]

This article has been cited by other articles:

				S. Kitaoka, T. Furuyashiki, A. Nishi, T. Shuto, S. Koyasu, T. Matsuoka, M. Miyasaka, P. Greengard, and S. Narumiya Prostaglandin E2 Acts on EP1 Receptor and Amplifies Both Dopamine D1 and D2 Receptor Signaling in the Striatum J. Neurosci., November 21, 2007; 27(47): 12900 - 12907. [Abstract] [Full Text] [PDF]

				T. Strahl, B. Grafelmann, J. Dannenberg, J. Thorner, and O. Pongs Conservation of Regulatory Function in Calcium-binding Proteins: HUMAN FREQUENIN (NEURONAL CALCIUM SENSOR-1) ASSOCIATES PRODUCTIVELY WITH YEAST PHOSPHATIDYLINOSITOL 4-KINASE ISOFORM, Pik1 J. Biol. Chem., December 5, 2003; 278(49): 49589 - 49599. [Abstract] [Full Text] [PDF]

				M. Rajebhosale, S. Greenwood, J. Vidugiriene, A. Jeromin, and S. Hilfiker Phosphatidylinositol 4-OH Kinase Is a Downstream Target of Neuronal Calcium Sensor-1 in Enhancing Exocytosis in Neuroendocrine Cells J. Biol. Chem., February 14, 2003; 278(8): 6075 - 6084. [Abstract] [Full Text] [PDF]

				C. S. Pao and J. L. Benovic Phosphorylation-Independent Desensitization of G Protein-Coupled Receptors? Sci. Signal., October 8, 2002; 2002(153): pe42 - pe42. [Abstract] [Full Text] [PDF]

				N. Kabbani, L. Negyessy, R. Lin, P. Goldman-Rakic, and R. Levenson Interaction with Neuronal Calcium Sensor NCS-1 Mediates Desensitization of the D2 Dopamine Receptor J. Neurosci., October 1, 2002; 22(19): 8476 - 8486. [Abstract] [Full Text] [PDF]

				S. Koizumi, P. Rosa, G. B. Willars, R. A. J. Challiss, E. Taverna, M. Francolini, M. D. Bootman, P. Lipp, K. Inoue, J. Roder, et al. Mechanisms Underlying the Neuronal Calcium Sensor-1-evoked Enhancement of Exocytosis in PC12 Cells J. Biol. Chem., August 9, 2002; 277(33): 30315 - 30324. [Abstract] [Full Text] [PDF]

				P Nokelainen and J Flint Genetic effects on human cognition: lessons from the study of mental retardation syndromes J. Neurol. Neurosurg. Psychiatry, March 1, 2002; 72(3): 287 - 296. [Abstract] [Full Text] [PDF]

				G. J. Mizejewski Alpha-fetoprotein Structure and Function: Relevance to Isoforms, Epitopes, and Conformational Variants Experimental Biology and Medicine, May 1, 2001; 226(5): 377 - 408. [Abstract] [Full Text]

				S. S. G. Ferguson Evolving Concepts in G Protein-Coupled Receptor Endocytosis: The Role in Receptor Desensitization and Signaling Pharmacol. Rev., March 1, 2001; 53(1): 1 - 24. [Abstract] [Full Text] [PDF]

				M. SALLESE, L. SALVATORE, E. D’URBANO, G. SALA, M. STORTO, T. LAUNEY, F. NICOLETTI, T. KNÖPFEL, and A. DE BLASI The G-protein-coupled receptor kinase GRK4 mediates homologous desensitization of metabotropic glutamate receptor 1 FASEB J, December 1, 2000; 14(15): 2569 - 2580. [Abstract] [Full Text]

				K.-N. Kim, Y. H. Cheong, R. Gupta, and S. Luan Interaction Specificity of Arabidopsis Calcineurin B-Like Calcium Sensors and Their Target Kinases Plant Physiology, December 1, 2000; 124(4): 1844 - 1853. [Abstract] [Full Text]

				M. Sallese, S. Mariggiò, E. D'Urbano, L. Iacovelli, and A. De Blasi Selective Regulation of Gq Signaling by G Protein-Coupled Receptor Kinase 2: Direct Interaction of Kinase N Terminus with Activated Galpha q Mol. Pharmacol., April 1, 2000; 57(4): 826 - 831. [Abstract] [Full Text]

				J. Shi, K.-N. Kim, O. Ritz, V. Albrecht, R. Gupta, K. Harter, S. Luan, and J. Kudla Novel Protein Kinases Associated with Calcineurin B-like Calcium Sensors in Arabidopsis PLANT CELL, December 1, 1999; 11(12): 2393 - 2406. [Abstract] [Full Text]

				L. Iacovelli, R. Franchetti, D. Grisolia, and A. De Blasi Selective Regulation of G Protein-Coupled Receptor-Mediated Signaling by G Protein-Coupled Receptor Kinase 2 in FRTL-5 Cells: Analysis of Thyrotropin, alpha 1B-Adrenergic, and A1 Adenosine Receptor-Mediated Responses Mol. Pharmacol., August 1, 1999; 56(2): 316 - 324. [Abstract] [Full Text]

				A. Ruiz-Gomez, J. Humrich, C. Murga, U. Quitterer, M. J. Lohse, and F. Mayor Jr. Phosphorylation of Phosducin and Phosducin-like Protein by G Protein-coupled Receptor Kinase 2 J. Biol. Chem., September 15, 2000; 275(38): 29724 - 29730. [Abstract] [Full Text] [PDF]

				K. Berrada, C. L. Plesnicher, X. Luo, and M. Thibonnier Dynamic Interaction of Human Vasopressin/Oxytocin Receptor Subtypes with G Protein-coupled Receptor Kinases and Protein Kinase C after Agonist Stimulation J. Biol. Chem., August 25, 2000; 275(35): 27229 - 27237. [Abstract] [Full Text] [PDF]

				N. Miwa, T. Uebi, and S. Kawamura Characterization of p26olf, a Novel Calcium-binding Protein in the Frog Olfactory Epithelium J. Biol. Chem., August 25, 2000; 275(35): 27245 - 27249. [Abstract] [Full Text] [PDF]

				M. M. Belcheva, M. Szucs, D. Wang, W. Sadee, and C. J. Coscia {micro}-Opioid Receptor-mediated ERK Activation Involves Calmodulin-dependent Epidermal Growth Factor Receptor Transactivation J. Biol. Chem., August 31, 2001; 276(36): 33847 - 33853. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Iacovelli, L. Articles by Blasi, A. D. Search for Related Content PubMed PubMed Citation Articles by Iacovelli, L.