Inhibition of G Protein-coupled Receptor Kinase Subtypes by Ca2+/Calmodulin
1996; Elsevier BV; Volume: 271; Issue: 45 Linguagem: Inglês
10.1074/jbc.271.45.28691
ISSN1083-351X
AutoresTsu Tshen Chuang, Lina Paolucci, Antonio De Blasi,
Tópico(s)Computational Drug Discovery Methods
ResumoG protein-coupled receptor kinases (GRKs) are implicated in the homologous desensitization of G protein-coupled receptors. Six GRK subtypes have so far been identified, named GRK1 to GRK6. The functional state of the GRKs can be actively regulated in different ways. In particular, it was found that retinal rhodopsin kinase (GRK1), but not the ubiquitous βARK1 (GRK2), can be inhibited by the photoreceptor-specific Ca2+-binding protein recoverin through direct binding. The present study was aimed to investigate regulation of other GRKs by alternative Ca2+-binding proteins such as calmodulin (CaM). We found that Gβγ-activated GRK2 and GRK3 were inhibited by CaM to similar extents (IC50∼ 2 μM), while a 50-fold more potent inhibitory effect was observed on GRK5 (IC50 = 40 nM). Inhibition by CaM was strictly dependent on Ca2+ and was prevented by the CaM inhibitor CaMBd. Since Gβγ, which is a binding target of Ca2+/CaM, is critical for the activation of GRK2 and GRK3, it provides a possible site of interaction between these proteins. However, since GRK5 is Gβγ-independent, an alternative mechanism is conceivable. A direct interaction between GRK5 and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. This binding does not influence the catalytic activity as demonstrated using the soluble GRK substrate casein. Instead, Ca2+/CaM significantly reduced GRK5 binding to the membrane. The mechanism of GRK5 inhibition appeared to be through direct binding to Ca2+/CaM, resulting in inhibition of membrane association and hence receptor phosphorylation. The present study provides the first evidence for a regulatory effect of Ca2+/CaM on some GRK subtypes, thus expanding the range of different mechanisms regulating the functional states of these kinases. G protein-coupled receptor kinases (GRKs) are implicated in the homologous desensitization of G protein-coupled receptors. Six GRK subtypes have so far been identified, named GRK1 to GRK6. The functional state of the GRKs can be actively regulated in different ways. In particular, it was found that retinal rhodopsin kinase (GRK1), but not the ubiquitous βARK1 (GRK2), can be inhibited by the photoreceptor-specific Ca2+-binding protein recoverin through direct binding. The present study was aimed to investigate regulation of other GRKs by alternative Ca2+-binding proteins such as calmodulin (CaM). We found that Gβγ-activated GRK2 and GRK3 were inhibited by CaM to similar extents (IC50∼ 2 μM), while a 50-fold more potent inhibitory effect was observed on GRK5 (IC50 = 40 nM). Inhibition by CaM was strictly dependent on Ca2+ and was prevented by the CaM inhibitor CaMBd. Since Gβγ, which is a binding target of Ca2+/CaM, is critical for the activation of GRK2 and GRK3, it provides a possible site of interaction between these proteins. However, since GRK5 is Gβγ-independent, an alternative mechanism is conceivable. A direct interaction between GRK5 and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. This binding does not influence the catalytic activity as demonstrated using the soluble GRK substrate casein. Instead, Ca2+/CaM significantly reduced GRK5 binding to the membrane. The mechanism of GRK5 inhibition appeared to be through direct binding to Ca2+/CaM, resulting in inhibition of membrane association and hence receptor phosphorylation. The present study provides the first evidence for a regulatory effect of Ca2+/CaM on some GRK subtypes, thus expanding the range of different mechanisms regulating the functional states of these kinases. INTRODUCTIONG protein-coupled receptor kinases (GRKs) 1The abbreviations used are: GRKG protein-coupled receptor kinaseCaMcalmodulinCaMBdcalmodulin-inhibiting peptideGβγβγ subunits of heterotrimeric G proteinNCSneuronal calcium sensorsROSrod outer segment(s)TSHthyroid-stimulating hormonePAGEpolyacrylamide gel electrophoresis. form a family of serine/threonine kinases that are implicated in the homologous desensitization of G protein-coupled receptors. Six GRK subtypes have so far been identified, named GRK1 to GRK6 according to the order of their discovery (1Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (469) Google Scholar). Of these six subtypes, rhodopsin kinase corresponds to GRK1, βARK1 to GRK2, and βARK2 to GRK3. Based on sequence homology, these six GRK subtypes are classified into three subgroups (1Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (469) Google Scholar): GRK1 is alone in the first group, GRK2 and GRK3 form the second group, while GRK4, −5, and −6 constitute the third subgroup. With the exception of GRK1 and GRK4, which are specifically localized in retina and pineal gland (GRK1) and testis (GRK4), the other four subtypes are ubiquitous.In homologous desensitization of G protein-coupled receptors, the binding of an agonist to its receptor induces a complicity of events to result in phosphorylation of the agonist-bound receptor by GRK. The phosphorylated receptor would then display increased affinity for an arrestin protein (2Wilson C.J. Applebury M.L. Curr. Biol. 1993; 3: 683-686Abstract Full Text PDF PubMed Scopus (37) Google Scholar), the binding of which to the phosphorylated receptor prevents any further coupling between the receptor and G proteins, and thus the receptor is rendered desensitized (inactivated) (1Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (469) Google Scholar, 2Wilson C.J. Applebury M.L. Curr. Biol. 1993; 3: 683-686Abstract Full Text PDF PubMed Scopus (37) Google Scholar). A critical determinant factor for efficient receptor phosphorylation by GRK is the colocalization of the kinase and its receptor substrate on the plasma membrane. The mechanisms involved have been extensively studied. For GRK1, receptor activation causes farnesylation of the C-terminal tail of the kinase, facilitating its localization to the membrane (3Kuhn H. Biochemistry. 1978; 21: 4389-4395Crossref Scopus (218) Google Scholar, 4Inglese J. Glickman J.F. Lorenz W. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 1422-1425Abstract Full Text PDF PubMed Google Scholar). For GRK2 and GRK3, which are highly homologous, membrane localization is shown to be mediated by binding to the βγ subunits of heterotrimeric G proteins (Gβγ) via their pleckstrin homology domains (5Pitcher J.A. Inglese J. Higgins J.B. Arriza J.L. Casey P.J. Kim C. Benovic J.L. Kwatra M.M. Caron M.G. Lefkowitz R.J. Science. 1992; 257: 1264-1267Crossref PubMed Scopus (563) Google Scholar, 6Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar). Palmitoylation appears to be the mechanism for GRK4 and GRK6 (7Premont R.T. Macrae A.D. Stoffel R.H. Chung N. Pitcher J.A. Ambrose C. Inglese J. MacDonald M.E. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 6403-6410Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 8Stoffel R.H. Randall R.R. Premont R.T. Lefkowitz R.J. Inglese J. J. Biol. Chem. 1994; 269: 27791-27794Abstract Full Text PDF PubMed Google Scholar), while electrostatic bonds between the basic C-terminal domain of the kinase and acidic phospholipids in the membrane are hypothesized to mediate membrane localization of GRK5 (1Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (469) Google Scholar, 9Kunapuli P. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1994; 269: 10209-10212Abstract Full Text PDF PubMed Google Scholar).The functional state of the GRK/arrestin machinery can be actively regulated via intracellular messenger pathways in different ways. These include changes in kinase activity through covalent modification (10Chuang T.T. LeVine III, H. De Blasi A. J. Biol. Chem. 1995; 270: 18660-18665Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) and/or modifications in their expression levels (11De Blasi A. Parruti G. Sallese M. J. Clin. Invest. 1995; 95: 203-210Crossref PubMed Google Scholar, 12Parruti G. Peracchia F. Sallese M. Ambrosini G. Masini M. Rotilio D. De Blasi A. J. Biol. Chem. 1993; 268: 9753-9761Abstract Full Text PDF PubMed Google Scholar). Such alterations result in modified potency of receptor desensitization and subsequently changes in receptor-mediated functions, as exemplified by the effects of GRK2 overexpression or inhibition on myocardial function in transgenic mice (13Koch W.J. Rockman H.A. Samama P. Hamilton R.A. Bond R.A. Milano C.A. Lefkowitz R.J. Science. 1995; 268: 1350-1353Crossref PubMed Scopus (635) Google Scholar). More recently, permanent overexpression of β-arrestin 1 in FRTL5 cells has been shown to inhibit the mitogenic activity of thyroid-stimulating hormone (TSH) (14Iacovelli L. Franchetti R. Masini M. De Blasi A. Mol. Endocrinol. 1996; 10: 1138-1146PubMed Google Scholar).In some settings regulation of GRK can also be mediated by Ca2+-binding proteins. It has been recently demonstrated that in the presence of Ca2+, GRK1 is inhibited by the photoreceptor-specific recoverin through direct binding (15Chen C.-K. Inglese J. Lefkowitz R.J. Hurley J.B. J. Biol. Chem. 1995; 270: 18060-18066Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 16Klenchin V.A. Calvert P.D. Bownds M.D. J. Biol. Chem. 1995; 270: 16147-16152Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Recoverin is a member of the family of neuron-specific proteins named neuronal calcium sensors (NCS); several other members of this family are also able to inhibit GRK1 in a calcium-dependent manner (17De Castro E. Nef S. Fiumelli H. Lenz S.E. Kawamura S. Nef P. Biochem. Biophys. Res. Commun. 1995; 216: 133-140Crossref PubMed Scopus (77) Google Scholar). Inhibition by recoverin does not appear to be a general phenomenon for the GRK family since it has no inhibitory activity on GRK2 (15Chen C.-K. Inglese J. Lefkowitz R.J. Hurley J.B. J. Biol. Chem. 1995; 270: 18060-18066Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). However, regulation of other GRK subgroups by alternative Ca2+-binding proteins cannot be discounted. In particular, the ubiquitous calcium-binding protein calmodulin (CaM) binds Gβγ in a Ca2+-dependent manner (18Katada T. Kusakabe K. Oinuma M. Ui M. J. Biol. Chem. 1987; 262: 11897-11900Abstract Full Text PDF PubMed Google Scholar, 19Mangels L.A. Neubig R.R. Hamm H.E. Gnegy M.E. Biochem. J. 1992; 283: 683-690Crossref PubMed Scopus (8) Google Scholar) and can therefore be envisaged to compete with GRK2 and GRK3 for Gβγ, resulting in inhibition of their activity.As an extension to our work on intracellular regulation of GRK (10Chuang T.T. LeVine III, H. De Blasi A. J. Biol. Chem. 1995; 270: 18660-18665Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 11De Blasi A. Parruti G. Sallese M. J. Clin. Invest. 1995; 95: 203-210Crossref PubMed Google Scholar, 12Parruti G. Peracchia F. Sallese M. Ambrosini G. Masini M. Rotilio D. De Blasi A. J. Biol. Chem. 1993; 268: 9753-9761Abstract Full Text PDF PubMed Google Scholar), we conducted the present study to test this hypothesis. It was observed that while Ca2+/CaM did inhibit GRK2 and GRK3, it demonstrated a dramatically greater potency in inhibiting GRK5 through direct binding to the kinase.RESULTSTo test the possibility that CaM may inhibit GRK2 activity by virtue of its ability to bind Gβγ in a Ca2+-dependent manner, the effect of CaM on GRK2 activity was examined in a Gβγ-dependent ROS phosphorylation assay (see "Experimental Procedures"). In the absence of Ca2+, CaM at 1 and 20 μM had little or no effect on GRK2, but when Ca2+ (1 mM) was present, the same concentrations of CaM resulted in pronounced inhibition of the Gβγ-mediated GRK2 activity (Fig. 1A). The IC50 value for this inhibitory action was approximately 2 μM. This Ca2+-dependent inhibition was proven to be CaM-dependent since it was completely reverted by the CaM-inhibiting peptide (CaMBd) (24Payne M.E. Fong Y.-L. Ono T. Colbran R.J. Kemp B.E. Soderling T.R. Means A.R. J. Biol. Chem. 1988; 263: 7190-7195Abstract Full Text PDF PubMed Google Scholar) derived from the CaM-dependent protein kinase II (Fig. 1B).Of the six GRKs, Gβγ has been shown to bind and activate only GRK2 and GRK3 through binding to their respective pleckstrin homology domains (5Pitcher J.A. Inglese J. Higgins J.B. Arriza J.L. Casey P.J. Kim C. Benovic J.L. Kwatra M.M. Caron M.G. Lefkowitz R.J. Science. 1992; 257: 1264-1267Crossref PubMed Scopus (563) Google Scholar, 6Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar). In an attempt to verify that this Ca2+/CaM inhibitory effect was dependent on its Gβγ binding activity, experiments were conducted to compare the effects of Ca2+/CaM on GRK2 and GRK3, which are dependent on Gβγ, with those on GRK1 and GRK5, which are Gβγ-insensitive. In the presence of 400 nM CaM plus Ca2+, both GRK2 and GRK3 were inhibited to similar extents, GRK1 was unaffected, but surprisingly GRK5 was dramatically inhibited (Fig. 2). Ca2+ alone had no effect on any of these four GRKs. Fig. 3A shows the dose-response effects of Ca2+/CaM on these four GRKs. Since a near maximal inhibition of GRK5 was achieved with 400 nM CaM, a detailed dose response of lower doses of CaM was performed (Fig. 3B). The inhibitory activity of CaM on GRK5 was ∼ 50-fold more potent than on GRK2, with an IC50 value of ∼ 40 nM. The effective concentration of CaM on GRK5 lies in the low range of CaM concentrations that have been shown to regulate target proteins (25O'Neil K.T. DeGrado W.F. Trends Biochem. Sci. 1990; 15: 59-64Abstract Full Text PDF PubMed Scopus (712) Google Scholar), and it is more potent than the inhibitory effect of Ca2+/recoverin on GRK1 (15Chen C.-K. Inglese J. Lefkowitz R.J. Hurley J.B. J. Biol. Chem. 1995; 270: 18060-18066Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 16Klenchin V.A. Calvert P.D. Bownds M.D. J. Biol. Chem. 1995; 270: 16147-16152Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). This is suggestive of a highly relevant interaction and had therefore channeled our interest into further experiments on this phenomenon.Fig. 2The inhibitory effect of Ca2+/calmodulin is selective for different GRK subtypes. GRK1 purified from ROS membranes and recombinant purified GRK2, GRK3, and GRK5 were incubated under phosphorylating conditions with urea-treated ROS. The level of phosphorylation found in the presence of Ca2+/CaM (400 nM) is expressed as % of control (i.e. in the absence of Ca/CaM). Gβγ (100 nM) was included in the assays of GRK2 and GRK3. Data are means ± S.E. from three experiments. Rhodopsin phosphorylation by different GRKs in the absence (−) or presence (+) of Ca2+/CaM from representative experiments is shown in the upper part of the figure.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Dose-dependent effect of Ca2+/calmodulin on different GRK subtypes. A, rhodopsin was phosphorylated by GRK1 (filled circles), GRK2 (filled squares), GRK3 (open circles), and GRK5 (open squares) in the presence of different concentrations of CaM. As the inhibition by GRK5 was almost complete at 400 nM CaM, a detailed dose response of lower doses of CaM was performed (B). Data are means ± S.E. (n = 3) or means of two experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)As for GRK2, the inhibition of GRK5 by Ca2+/CaM was Ca2+-dependent (Fig. 4A) and was almost completely reverted by the CaM-inhibiting peptide CaMBd (Fig. 4B). These results confirmed that the inhibitory effect was mediated by CaM. The inhibition was effectively reverted by increasing the concentration of GRK5 in the phosphorylation reaction by 40-fold (Fig. 5).Fig. 4Characterization of the effect of Ca2+/calmodulin on GRK5. A, rhodopsin phosphorylation by GRK5 in the presence of 400 nM CaM ± 1 mM Ca2+. B, the effect of Ca2+/CaM was prevented by CaM inhibitor CaMBd in a dose-dependent manner. The arrow indicates bands of phosphorylated rhodopsin (Opsin) as revealed by autoradiography after polyacrylamide gel electophoresis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Reversal of Ca2+/calmodulin inhibition by increasing concentrations of GRK5. Rhodopsin was incubated with the indicated concentrations of GRK5 in the absence or presence of Ca2+/CaM (CaM = 100 nM) (means of two experiments). The autoradiography from one representative experiment is shown in the upper part of the figure. Because of the different amounts of GRK5, the exposure of autoradiography was for 2.5 h (for 5 nM GRK5) or 10 min (for 50 and 200 nM GRK5).View Large Image Figure ViewerDownload Hi-res image Download (PPT)One mechanism by which Ca2+/CaM could inhibit GRK5 was by direct binding to GRK5, as in the case of GRK1 inhibition by Ca2+/recoverin (15Chen C.-K. Inglese J. Lefkowitz R.J. Hurley J.B. J. Biol. Chem. 1995; 270: 18060-18066Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). To address this possibility, in vitro binding assays were performed using CaM-conjugated Sepharose 4B gel and unconjugated Sepharose 4B as negative control (Fig. 6). Gβγ was used as a positive control for the binding assay since it has previously been shown to bind CaM-Sepharose 4B in a Ca2+-dependent manner (18Katada T. Kusakabe K. Oinuma M. Ui M. J. Biol. Chem. 1987; 262: 11897-11900Abstract Full Text PDF PubMed Google Scholar, 19Mangels L.A. Neubig R.R. Hamm H.E. Gnegy M.E. Biochem. J. 1992; 283: 683-690Crossref PubMed Scopus (8) Google Scholar). Both GRK5 and Gβγ bound to CaM-Sepharose 4B in a Ca2+-dependent manner, though trace amounts of both also bound in the absence of Ca2+. Densitometric analysis showed that the -fold excess of CaM-Sepharose-bound protein/control-Sepharose-bound protein was 5.8 and 4.3 for GRK5 and Gβγ, respectively (n = 2). Neither of them bound to the unconjugated Sepharose 4B gel. Similar results were obtained when the molarity ratio between CaM and the ligand was varied by 3-fold (CaM, ligand = 60 or 180:1). These binding assays document a direct, Ca2+-dependent interaction between GRK5 and CaM. Unlike GRK5, GRK2 (30, 100, and 200 nM) did not bind to CaM-conjugated Sepharose 4B gel (data not shown), supporting the specificity of this interaction.Fig. 6Direct binding of GRK5 and Gβγ to calmodulin. Sepharose 4B-conjugated CaM (CaMSg) was incubated (4°C for 1 h) with 100 nM GRK5 (top) or 100 nM Gβγ purified from bovine brain (bottom) in the presence or absence of Ca2+. Sepharose 4B without conjugated CaM (Sg) was used as negative control. Bound GRK5 or Gβγ separated by centrifugation followed by extensive washing were incubated in SDS sample buffer at 95°C for 5 min, run on 10% SDS-PAGE, and blotted onto nitrocellulose paper. Bound GRK5 or Gβγ were revealed by specific antibodies (arrows). The experiment shown is representative of three similar experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Similar to other kinases, GRK5 contains catalytic and regulatory domains. Binding of Ca2+/CaM to GRK5 may result in inhibition of either of these domains. To distinguish between these two possibilities, the phosphorylation assays were repeated in a membrane-free system using the soluble protein casein instead of ROS as substrate (26Pitcher J.A. Touhara K. Payne E.S. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 11707-11710Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). The results shown in Fig. 7 demonstrate clearly the lack of any inhibitory effect on casein phosphorylation by GRK5. This suggested that the effect of Ca2+/CaM would most likely be exerted on non-catalytic domains, which have regulatory roles.Fig. 7Effect of Ca2+/calmodulin on ROS and casein phosphorylation by GRK5. Urea-treated ROS (left) or casein (10 μM, right) was incubated with GRK5 under phosphorylating conditions in the presence or absence of Ca2+/CaM. The arrows indicate bands of phosphorylated rhodopsin (Opsin) and casein as revealed by autoradiography after polyacrylamide gel electophoresis. The higher molecular weight phosphorylated protein observed on the left panel, but not on the right panel, and also in Fig. 4A likely represents opsin dimer (36Benovic J.L. Mayor Jr., F. Somers R. Caron M. Lefkowitz R.J. Nature. 1986; 321: 869-872Crossref PubMed Scopus (135) Google Scholar). The experiment is representative of two similar experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Since membrane localization is a prerequisite for efficient receptor phosphorylation by all GRKs, the effect of Ca2+/CaM on the binding of GRK5 to ROS membranes was examined using a centrifugation assay. GRK5 and CaM ± Ca2+ were incubated together prior to the initiation of phosphorylation reaction by the addition of ROS and ATP. At the end of the reaction, ROS membrane fraction was separated from the soluble fraction by centrifugation, the quantities of membrane-bound GRK5 were analyzed by Western blotting, and rhodopsin phosphorylation was revealed by autoradiography. In the absence of Ca2+, the quantity of GRK5 bound to the membrane was unaffected by CaM. When GRK5 was exposed to CaM in the presence of Ca2+, a dramatic reduction in GRK5 translocation onto the membrane was observed (Fig. 8). Densitometric analysis showed a 10-fold lower amount of GRK5 bound to the membrane when in the presence of Ca2+/CaM. This Ca2+-dependent inhibition of GRK5 translocation was paralleled by the effects of CaM on rhodopsin phosphorylation activity by the same samples (Fig. 8).Fig. 8Binding of GRK5 to ROS membranes is inhibited by Ca2+/calmodulin. Urea-treated ROS was incubated (30°C for 30 min) with 400 nM GRK5 under phosphorylating conditions in the presence or absence of Ca2+ and/or CaM (5 μM). At the end of the incubation, the samples were chilled and centrifuged (48,000 × g for 30 min at 4°C) to pellet membranes. The pellet was resuspended in SDS sample buffer and divided into two aliquots. One aliquot was run on 10% SDS-polyacrylamide gel and blotted onto nitrocellulose paper, and membrane-bound GRK5 was revealed by specific antibodies (top panel). The second aliquot (lower panel) was run on 10% SDS-PAGE, and autoradiography of the dry gel was done to reveal phosphorylated rhodopsin (arrows). The experiment shown is representative of two similar experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONThe present study demonstrates for the first time Ca2+-dependent inhibitory effects of CaM on some members of the GRK family. The Gβγ-activated GRK2 and GRK3 were inhibited to similar extents, while a 50-fold more potent inhibitory effect was observed on GRK5. The mechanism of GRK5 inhibition appeared to be through direct binding to Ca2+/CaM, resulting in inhibition of membrane association and hence receptor phosphorylation.CaM is an acidic protein that is considered the primary "decoder" of Ca2+ information in the cell (25O'Neil K.T. DeGrado W.F. Trends Biochem. Sci. 1990; 15: 59-64Abstract Full Text PDF PubMed Scopus (712) Google Scholar, 27James P. Vorherr T. Carafoli E. Trends Biochem. Sci. 1995; 20: 38-42Abstract Full Text PDF PubMed Scopus (346) Google Scholar), exerting many of its functions when bound to Ca2+ (4 Ca2+ ions per CaM molecule). Numerous proteins have been identified to be regulated by Ca2+/CaM, e.g. kinases, phosphodiesterases, calcium pumps, and adenylate cyclase. Gβγ is also a binding target of Ca2+/CaM, as demonstrated previously (18Katada T. Kusakabe K. Oinuma M. Ui M. J. Biol. Chem. 1987; 262: 11897-11900Abstract Full Text PDF PubMed Google Scholar, 19Mangels L.A. Neubig R.R. Hamm H.E. Gnegy M.E. Biochem. J. 1992; 283: 683-690Crossref PubMed Scopus (8) Google Scholar) and confirmed in the present study. More recently, it has been established that the binding of GRK2 and GRK3 to Gβγ is critical in mediating the activation of these two GRK subtypes (5Pitcher J.A. Inglese J. Higgins J.B. Arriza J.L. Casey P.J. Kim C. Benovic J.L. Kwatra M.M. Caron M.G. Lefkowitz R.J. Science. 1992; 257: 1264-1267Crossref PubMed Scopus (563) Google Scholar, 6Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar). Therefore, Gβγ provides a possible site of indirect interaction between GRK2 and GRK3 with Ca2+/CaM. Indeed, in the present study, using a receptor phosphorylation assay in which the Gβγ-activated GRK2 and GRK3 activity was measured, we observed inhibition of GRK activity by CaM in a Ca2+-dependent manner. This may be due to blockade of Gβγ by Ca2+/CaM, thus inhibiting GRK activity. However, this does not preclude the possibility of Ca2+/CaM inhibiting the activity of GRK2 and GRK3 through alternative targets important in the functioning of these kinases.The IC50 value of CaM for GRK2 is 2 μM. This is within the reported range of cellular CaM levels of 1-10 μM (28Gnegy M.E. Annu. Rev. Pharmacol. Toxicol. 1993; 33: 45-70Crossref PubMed Google Scholar) and is similar to the IC50 value of Ca2+/recoverin inhibition of GRK1 (15Chen C.-K. Inglese J. Lefkowitz R.J. Hurley J.B. J. Biol. Chem. 1995; 270: 18060-18066Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), thus suggesting a possible physiological implication for the functional interaction between CaM and GRK2, possibly through their common binding target of Gβγ.The more significant finding of the present study was the unexpected observation of a potent inhibitory effect of Ca2+/CaM on GRK5. Similar to GRK2 and GRK3, GRK5 inhibition by CaM was strictly dependent on Ca2+ and was prevented by the CaM inhibitor CaMBd. However, since GRK5 activity is not influenced by Gβγ (21Premont R.T. Koch W.J. Inglese J. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6832-6841Abstract Full Text PDF PubMed Google Scholar, 29Kunapuli P. Benovic J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5588-5592Crossref PubMed Scopus (117) Google Scholar), there must be an alternative site of interaction between GRK5 and CaM. This site does not appear to be rhodopsin since Ca2+/CaM was at least 50-fold more potent in inhibiting ROS phosphorylation by GRK5 than by GRK2 and GRK3 and had no effects on GRK1. The affinity of these GRKs for rhodopsin is very similar, and Km values are 4, 6, and 2.2 μM for GRK1, −2, and −5, respectively (21Premont R.T. Koch W.J. Inglese J. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6832-6841Abstract Full Text PDF PubMed Google Scholar, 30Palczewski K. Benovic J.L. Trends Biochem. Sci. 1991; 16: 387-391Abstract Full Text PDF PubMed Scopus (185) Google Scholar).A direct interaction between GRK5 and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. The Ca2+/CaM binding site on GRK5 is as yet unidentified, but it is apparent that this binding does not influence the catalytic activity of the kinase as demonstrated by the lack of inhibitory effect on its phosphorylating activity on the soluble GRK substrate casein. Instead, Ca2+/CaM significantly reduced GRK5 binding to the membrane. Kunapuli et al. (9Kunapuli P. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1994; 269: 10209-10212Abstract Full Text PDF PubMed Google Scholar) 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. Therefore, similar to GRK1, −2, and −3, membrane anchoring of GRK5 is also a critical step in leading to receptor phosphorylation.In a recent report, six members of the NCS protein family have been shown to inhibit GRK1 phosphorylation of rhodopsin in a Ca2+-dependent manner (17De Castro E. Nef S. Fiumelli H. Lenz S.E. Kawamura S. Nef P. Biochem. Biophys. Res. Commun. 1995; 216: 133-140Crossref PubMed Scopus (77) Google Scholar). The authors also suggested a possible interaction between NCS and GRK5. While this possibility remains to be tested, other reports have demonstrated that the brain is not a major site of expression for this GRK subtype (21Premont R.T. Koch W.J. Inglese J. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6832-6841Abstract Full Text PDF PubMed Google Scholar, 29Kunapuli P. Benovic J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5588-5592Crossref PubMed Scopus (117) Google Scholar). This incompatibility between the sites of expression of GRK5 and NCS would argue against a physiological interaction between them. On the other hand, colocalization of GRK5 and CaM is quite possible due to the ubiquity of the latter protein. Since CaM and NCS are all calcium sensors, the present study suggests that they act as functional analogues in mediating the regulation of different GRK subtypes by Ca2+. Together, they are the first endogenous GRK inhibitors demonstrated so far.A number of G protein-coupled receptors are substrates of GRK2, −3, and −5, and some of these receptors are coupled to fluctuations in intracellular calcium concentrations, e.g. substance P and angiotensin II receptors (20Kwatra M.M. Schwinn D.A. Schreurs J. Blank J.L. Kim C.M. Benovic J.L. Krause J.E. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 9161-9164Abstract Full Text PDF PubMed Google Scholar, 31Oppermann M. Freedman N.J. Alexander R.W. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Ca2+/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 Ca2+/CaM may therefore mediate a novel route of cross-talk 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. There are examples of this nature (see Ref. 28Gnegy M.E. Annu. Rev. Pharmacol. Toxicol. 1993; 33: 45-70Crossref PubMed Google Scholar for review). Some of these reported synergistic interaction between CaM and hormonal activation of adenylyl cyclase. Of particular interest to GRK5 is the potentiation by CaM of TSH-stimulated adenylyl cyclase in the human thyroid gland (32Lakey T. MacNeil S. Humphreys H. Walker S.W. Munro D.S. Tomlinson S. Biochem. J. 1985; 225: 581-589Crossref PubMed Scopus (22) Google Scholar). Desensitization of the TSH receptor has recently been shown to be mediated by GRK5 (33Nagayama Y. Tanaka K. Hara T. Namba H. Yamashita S. Taniyama K. Niwa M. J. Biol. Chem. 1996; 271: 10143-10148Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). It is therefore conceivable that CaM inhibition of GRK5 contributes to its potentiating effect on TSH stimulation (32Lakey T. MacNeil S. Humphreys H. Walker S.W. Munro D.S. Tomlinson S. Biochem. J. 1985; 225: 581-589Crossref PubMed Scopus (22) Google Scholar) in addition to other effects of CaM on the adenylyl cyclase-cAMP axis, such as activation of the cyclase (28Gnegy M.E. Annu. Rev. Pharmacol. Toxicol. 1993; 33: 45-70Crossref PubMed Google Scholar). Another study stressed the importance of temporal overlapping of the Ca2+ pulse with the addition of the facilitatory neurotransmitter serotonin in classical conditioning in Aplysia (34Abrams T.W. Karl K.A. Kandel E.R. J. Neurosci. 1991; 11: 2655-2665Crossref PubMed Google Scholar). For the adrenocorticotrophic hormone receptor, the CaM-mediated potentiation effect was attributed to an action on receptor-Gs coupling (35Papadopoulos V. Widmaier E.P. Hall P.F. Endocrinology. 1990; 126: 2465-2473Crossref PubMed Scopus (19) Google Scholar), which is the classical site of intervention by GRKs. Such synergistic effects of CaM on the functions of receptors may be at least partially mediated by the effect of Ca2+/CaM on GRKs.In conclusion, the present study provides the first evidence for a regulatory effect of Ca2+/CaM on some GRK subtypes, thus expanding the range of different mechanisms by which the functional states of GRK/arrestin proteins can be regulated. The effect of Ca2+-sensor proteins appears to be rather general, and the GRK subtype selectivity documented for recoverin and CaM indicates that different kinases can be specifically regulated in different target tissues. INTRODUCTIONG protein-coupled receptor kinases (GRKs) 1The abbreviations used are: GRKG protein-coupled receptor kinaseCaMcalmodulinCaMBdcalmodulin-inhibiting peptideGβγβγ subunits of heterotrimeric G proteinNCSneuronal calcium sensorsROSrod outer segment(s)TSHthyroid-stimulating hormonePAGEpolyacrylamide gel electrophoresis. form a family of serine/threonine kinases that are implicated in the homologous desensitization of G protein-coupled receptors. Six GRK subtypes have so far been identified, named GRK1 to GRK6 according to the order of their discovery (1Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (469) Google Scholar). Of these six subtypes, rhodopsin kinase corresponds to GRK1, βARK1 to GRK2, and βARK2 to GRK3. Based on sequence homology, these six GRK subtypes are classified into three subgroups (1Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (469) Google Scholar): GRK1 is alone in the first group, GRK2 and GRK3 form the second group, while GRK4, −5, and −6 constitute the third subgroup. With the exception of GRK1 and GRK4, which are specifically localized in retina and pineal gland (GRK1) and testis (GRK4), the other four subtypes are ubiquitous.In homologous desensitization of G protein-coupled receptors, the binding of an agonist to its receptor induces a complicity of events to result in phosphorylation of the agonist-bound receptor by GRK. The phosphorylated receptor would then display increased affinity for an arrestin protein (2Wilson C.J. Applebury M.L. Curr. Biol. 1993; 3: 683-686Abstract Full Text PDF PubMed Scopus (37) Google Scholar), the binding of which to the phosphorylated receptor prevents any further coupling between the receptor and G proteins, and thus the receptor is rendered desensitized (inactivated) (1Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (469) Google Scholar, 2Wilson C.J. Applebury M.L. Curr. Biol. 1993; 3: 683-686Abstract Full Text PDF PubMed Scopus (37) Google Scholar). A critical determinant factor for efficient receptor phosphorylation by GRK is the colocalization of the kinase and its receptor substrate on the plasma membrane. The mechanisms involved have been extensively studied. For GRK1, receptor activation causes farnesylation of the C-terminal tail of the kinase, facilitating its localization to the membrane (3Kuhn H. Biochemistry. 1978; 21: 4389-4395Crossref Scopus (218) Google Scholar, 4Inglese J. Glickman J.F. Lorenz W. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 1422-1425Abstract Full Text PDF PubMed Google Scholar). For GRK2 and GRK3, which are highly homologous, membrane localization is shown to be mediated by binding to the βγ subunits of heterotrimeric G proteins (Gβγ) via their pleckstrin homology domains (5Pitcher J.A. Inglese J. Higgins J.B. Arriza J.L. Casey P.J. Kim C. Benovic J.L. Kwatra M.M. Caron M.G. Lefkowitz R.J. Science. 1992; 257: 1264-1267Crossref PubMed Scopus (563) Google Scholar, 6Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar). Palmitoylation appears to be the mechanism for GRK4 and GRK6 (7Premont R.T. Macrae A.D. Stoffel R.H. Chung N. Pitcher J.A. Ambrose C. Inglese J. MacDonald M.E. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 6403-6410Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 8Stoffel R.H. Randall R.R. Premont R.T. Lefkowitz R.J. Inglese J. J. Biol. Chem. 1994; 269: 27791-27794Abstract Full Text PDF PubMed Google Scholar), while electrostatic bonds between the basic C-terminal domain of the kinase and acidic phospholipids in the membrane are hypothesized to mediate membrane localization of GRK5 (1Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (469) Google Scholar, 9Kunapuli P. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1994; 269: 10209-10212Abstract Full Text PDF PubMed Google Scholar).The functional state of the GRK/arrestin machinery can be actively regulated via intracellular messenger pathways in different ways. These include changes in kinase activity through covalent modification (10Chuang T.T. LeVine III, H. De Blasi A. J. Biol. Chem. 1995; 270: 18660-18665Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) and/or modifications in their expression levels (11De Blasi A. Parruti G. Sallese M. J. Clin. Invest. 1995; 95: 203-210Crossref PubMed Google Scholar, 12Parruti G. Peracchia F. Sallese M. Ambrosini G. Masini M. Rotilio D. De Blasi A. J. Biol. Chem. 1993; 268: 9753-9761Abstract Full Text PDF PubMed Google Scholar). Such alterations result in modified potency of receptor desensitization and subsequently changes in receptor-mediated functions, as exemplified by the effects of GRK2 overexpression or inhibition on myocardial function in transgenic mice (13Koch W.J. Rockman H.A. Samama P. Hamilton R.A. Bond R.A. Milano C.A. Lefkowitz R.J. Science. 1995; 268: 1350-1353Crossref PubMed Scopus (635) Google Scholar). More recently, permanent overexpression of β-arrestin 1 in FRTL5 cells has been shown to inhibit the mitogenic activity of thyroid-stimulating hormone (TSH) (14Iacovelli L. Franchetti R. Masini M. De Blasi A. Mol. Endocrinol. 1996; 10: 1138-1146PubMed Google Scholar).In some settings regulation of GRK can also be mediated by Ca2+-binding proteins. It has been recently demonstrated that in the presence of Ca2+, GRK1 is inhibited by the photoreceptor-specific recoverin through direct binding (15Chen C.-K. Inglese J. Lefkowitz R.J. Hurley J.B. J. Biol. Chem. 1995; 270: 18060-18066Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 16Klenchin V.A. Calvert P.D. Bownds M.D. J. Biol. Chem. 1995; 270: 16147-16152Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Recoverin is a member of the family of neuron-specific proteins named neuronal calcium sensors (NCS); several other members of this family are also able to inhibit GRK1 in a calcium-dependent manner (17De Castro E. Nef S. Fiumelli H. Lenz S.E. Kawamura S. Nef P. Biochem. Biophys. Res. Commun. 1995; 216: 133-140Crossref PubMed Scopus (77) Google Scholar). Inhibition by recoverin does not appear to be a general phenomenon for the GRK family since it has no inhibitory activity on GRK2 (15Chen C.-K. Inglese J. Lefkowitz R.J. Hurley J.B. J. Biol. Chem. 1995; 270: 18060-18066Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). However, regulation of other GRK subgroups by alternative Ca2+-binding proteins cannot be discounted. In particular, the ubiquitous calcium-binding protein calmodulin (CaM) binds Gβγ in a Ca2+-dependent manner (18Katada T. Kusakabe K. Oinuma M. Ui M. J. Biol. Chem. 1987; 262: 11897-11900Abstract Full Text PDF PubMed Google Scholar, 19Mangels L.A. Neubig R.R. Hamm H.E. Gnegy M.E. Biochem. J. 1992; 283: 683-690Crossref PubMed Scopus (8) Google Scholar) and can therefore be envisaged to compete with GRK2 and GRK3 for Gβγ, resulting in inhibition of their activity.As an extension to our work on intracellular regulation of GRK (10Chuang T.T. LeVine III, H. De Blasi A. J. Biol. Chem. 1995; 270: 18660-18665Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 11De Blasi A. Parruti G. Sallese M. J. Clin. Invest. 1995; 95: 203-210Crossref PubMed Google Scholar, 12Parruti G. Peracchia F. Sallese M. Ambrosini G. Masini M. Rotilio D. De Blasi A. J. Biol. Chem. 1993; 268: 9753-9761Abstract Full Text PDF PubMed Google Scholar), we conducted the present study to test this hypothesis. It was observed that while Ca2+/CaM did inhibit GRK2 and GRK3, it demonstrated a dramatically greater potency in inhibiting GRK5 through direct binding to the kinase.
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