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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. 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 be mediated by Ca2+-binding proteins. has been demonstrated that in the of GRK1 is inhibited by the photoreceptor-specific recoverin through direct binding Inglese J. Lefkowitz R.J. J.B. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). is a of the family of proteins named calcium other of family are to inhibit GRK1 in a H. P. 1995; PubMed Scopus Google Scholar). Inhibition by recoverin does not to be a for the GRK family since it has inhibitory activity on GRK2 Inglese J. Lefkowitz R.J. J.B. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). However, regulation of other GRK subgroups by alternative Ca2+-binding proteins be In particular, the ubiquitous protein calmodulin in a M. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R.R. M.E. J. 1992; PubMed Scopus Google Scholar) and can be to GRK2 and GRK3 for Gβγ, resulting in inhibition of their an to 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, 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), the present study to was observed that while Ca2+/CaM inhibit GRK2 and GRK3, it demonstrated a potency in GRK5 through direct binding to the the that CaM inhibit GRK2 activity by of its to in a the effect of CaM on GRK2 activity was in a phosphorylation In the of CaM 1 and or effect on GRK2, but Ca2+ was the of CaM in inhibition of the GRK2 activity The for inhibitory was 2 This inhibition was to be since it was by the M.E. R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) the protein kinase the six has been shown to and GRK2 and GRK3 through binding to 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). In an to that Ca2+/CaM inhibitory effect was dependent on its binding were to the effects of Ca2+/CaM on GRK2 and GRK3, which are dependent on Gβγ, on GRK1 and which are In the of CaM GRK2 and GRK3 were inhibited to similar GRK1 was but GRK5 was inhibited Ca2+ alone effect on any of these four the effects of Ca2+/CaM on these four Since a inhibition of GRK5 was a of of CaM was The inhibitory activity of CaM on GRK5 was 50-fold more potent on GRK2, an of 40 The of CaM on GRK5 in the range of CaM that have been shown to target proteins Full Text PDF PubMed Scopus Google Scholar), and it is more potent the inhibitory effect of on GRK1 Inglese J. Lefkowitz R.J. J.B. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). This is of a highly interaction and into further on inhibitory effect of is for different GRK GRK1 and GRK2, GRK3, and GRK5 were The of phosphorylation found in the of Ca2+/CaM is as of in the of was in the of GRK2 and GRK3. are three phosphorylation by different GRKs in the or of Ca2+/CaM is shown in the of the effect of on different GRK rhodopsin was phosphorylated by GRK1 GRK2 GRK3 and GRK5 in the of different of the inhibition by GRK5 was a of of CaM was are = or of for GRK2, the inhibition of GRK5 by Ca2+/CaM was and was by the These that the inhibitory effect was mediated by The inhibition was by the of GRK5 in the phosphorylation by of the effect of on rhodopsin phosphorylation by GRK5 in the of CaM 1 the effect of Ca2+/CaM was prevented by CaM inhibitor in a The of phosphorylated rhodopsin as revealed by gel of inhibition by of was the of GRK5 in the or of Ca2+/CaM = of The is shown in the of the of the different of the of was for or and mechanism by which Ca2+/CaM inhibit GRK5 was by direct binding to as in the of GRK1 inhibition by Inglese J. Lefkowitz R.J. J.B. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). in binding were using CaM-conjugated Sepharose gel and Sepharose as was used as a for the binding since it has been shown to in a M. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R.R. M.E. J. 1992; PubMed Scopus Google Scholar). GRK5 and to in a of in the of that the of protein was and for GRK5 and Gβγ, = of to the Sepharose were the between CaM and the was by = or These binding a interaction between GRK5 and GRK2 and not to CaM-conjugated Sepharose gel not the of binding of GRK5 and to Sepharose CaM was for 1 GRK5 or in the or of Sepharose CaM was used as GRK5 or by by were in for on and GRK5 or were revealed by The shown is of three similar to other GRK5 catalytic and regulatory of Ca2+/CaM to GRK5 result in inhibition of of these between these the phosphorylation were in a using the soluble protein of as substrate J.A. Lefkowitz R.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The shown in the of any inhibitory effect on phosphorylation by This that the effect of Ca2+/CaM would be on which have regulatory of on and phosphorylation by or was GRK5 in the or of The of phosphorylated rhodopsin and as revealed by gel The phosphorylated protein observed on the but not on the and in J.L. F. R. Caron M. Lefkowitz R.J. PubMed Scopus Google Scholar). The is of similar membrane localization is a for efficient receptor phosphorylation by the effect of Ca2+/CaM on the binding of GRK5 to was using a GRK5 and CaM Ca2+ were to the of phosphorylation by the of and the of the membrane was the soluble by the of GRK5 were by and rhodopsin phosphorylation was revealed by In the of the of GRK5 to the membrane was by GRK5 was to CaM in the of a in GRK5 the membrane was observed a of GRK5 to the membrane in the of This inhibition of GRK5 was by the effects of CaM on rhodopsin phosphorylation activity by the of GRK5 to is inhibited by was for GRK5 in the or of Ca2+ and/or CaM the of the the were and for to The was in and into was on gel and and GRK5 was revealed by The second was on and of the gel was to phosphorylated rhodopsin The shown is of similar present study for the first inhibitory effects of CaM on some of the GRK The Gβγ-activated GRK2 and GRK3 were inhibited to similar while a 50-fold more potent inhibitory effect was observed on The mechanism of GRK5 inhibition appeared to be through direct binding to Ca2+/CaM, resulting in inhibition of membrane association and hence receptor is an acidic protein that is the of Ca2+ in the Full Text PDF PubMed Scopus Google Scholar, P. 1995; Full Text PDF PubMed Scopus Google Scholar), of its to Ca2+ Ca2+ CaM proteins have been to be regulated by Ca2+/CaM, calcium and is a binding target of Ca2+/CaM, as demonstrated M. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R.R. M.E. J. 1992; PubMed Scopus Google Scholar) and in the present More recently, it has been that the binding of GRK2 and GRK3 to is critical in the activation of these 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). provides a possible site of interaction between GRK2 and GRK3 in the present using a receptor phosphorylation in which the Gβγ-activated GRK2 and GRK3 activity was observed inhibition of GRK activity by CaM in a This be to of by Ca2+/CaM, thus GRK However, does not the of Ca2+/CaM the activity of GRK2 and GRK3 through alternative in the of these of CaM for GRK2 is 2 This is the range of CaM levels of M.E. 1993; PubMed Google Scholar) and is similar to the of inhibition of GRK1 Inglese J. Lefkowitz R.J. J.B. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), thus a possible for the functional interaction between CaM and GRK2, through their binding target of more of the present study was the of a potent inhibitory effect of Ca2+/CaM on 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 by R.T. W.J. Inglese J. Lefkowitz R.J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, P. Benovic J.L. A. 1993; PubMed Scopus Google Scholar), be an alternative site of interaction between GRK5 and This site does not to be rhodopsin since Ca2+/CaM was 50-fold more potent in phosphorylation by GRK5 by GRK2 and GRK3 and effects on The affinity of these GRKs for rhodopsin is and are and for GRK1, and −5, R.T. W.J. Inglese J. Lefkowitz R.J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, Benovic J.L. Full Text PDF PubMed Scopus Google direct interaction between GRK5 and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. The Ca2+/CaM binding site on GRK5 is as but it is that binding does not influence the catalytic activity of the kinase as demonstrated by the of inhibitory effect on its activity on the soluble GRK substrate casein. Instead, Ca2+/CaM significantly reduced GRK5 binding to the membrane. 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 the inhibition of GRK5 association the membrane by Ca2+/CaM would kinase hence the observed inhibitory effect on receptor phosphorylation. similar to GRK1, and membrane of GRK5 is a critical in to receptor a six of the protein family have been shown to inhibit GRK1 phosphorylation of rhodopsin in a H. P. 1995; PubMed Scopus Google Scholar). The a possible interaction between and to be other have demonstrated that the is not a site of expression for GRK R.T. W.J. Inglese J. Lefkowitz R.J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, P. Benovic J.L. A. 1993; PubMed Scopus Google Scholar). This between the of expression of GRK5 and would a interaction between the other colocalization of GRK5 and CaM is possible to the of the Since CaM and are calcium the present study that as functional in the regulation of different GRK subtypes by are the first GRK demonstrated so of G protein-coupled are of GRK2, and −5, and some of these are to in intracellular calcium and M.M. J. J.L. Kim Benovic J.L. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, M. Lefkowitz R.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). Ca2+/CaM can a mechanism for homologous desensitization of these receptors. in any a of receptor for GRK2, and be and Ca2+/CaM mediate a of in which receptor agonist can the activity of these GRK subtypes, thus the functional of a different are of M.E. 1993; PubMed Google for of these interaction between CaM and activation of Of to GRK5 is the by CaM of in the gland H. J. PubMed Scopus Google Scholar). of the receptor has been shown to be mediated by GRK5 H. M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). is that CaM inhibition of GRK5 to its effect on H. J. PubMed Scopus Google Scholar) in to other effects of CaM on the such as activation of the M.E. 1993; PubMed Google Scholar). study the of of the Ca2+ the of the in in J. PubMed Google Scholar). For the hormone the effect was to an on coupling PubMed Scopus Google Scholar), which is the site of by Such effects of CaM on the of be mediated by the effect of Ca2+/CaM on 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 The effect of proteins appears to be and the GRK for recoverin and CaM that different kinases can be specifically regulated in different target 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 be mediated by Ca2+-binding proteins. has been demonstrated that in the of GRK1 is inhibited by the photoreceptor-specific recoverin through direct binding Inglese J. Lefkowitz R.J. J.B. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). is a of the family of proteins named calcium other of family are to inhibit GRK1 in a H. P. 1995; PubMed Scopus Google Scholar). Inhibition by recoverin does not to be a for the GRK family since it has inhibitory activity on GRK2 Inglese J. Lefkowitz R.J. J.B. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). However, regulation of other GRK subgroups by alternative Ca2+-binding proteins be In particular, the ubiquitous protein calmodulin in a M. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R.R. M.E. J. 1992; PubMed Scopus Google Scholar) and can be to GRK2 and GRK3 for Gβγ, resulting in inhibition of their an to 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, 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), the present study to was observed that while Ca2+/CaM inhibit GRK2 and GRK3, it demonstrated a potency in GRK5 through direct binding to the
Chuang et al. (Fri,) studied this question.
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