Structure-Function Analysis of G Protein-coupled Receptor Kinase-5

Many G protein-coupled receptors are phosphorylated and regulated by a distinct family of G protein-coupled receptor kinases (GRKs) that specifically target the activated form of the receptor. Recent studies have revealed that the GRKs are also subject to post-translational regulation. For example, GRK5 activity is strongly inhibited by protein kinase C phosphorylation and by Ca2+-calmodulin binding. Ca2+-calmodulin binding also promotes GRK5 autophosphorylation, which further contributes to kinase inhibition. In this study we identify two important structural domains in GRK5, a phospholipid binding domain (residues 552–562) and an autoinhibitory domain (residues 563–590), that significantly contribute to GRK5 localization and function. We demonstrate that the C-terminal region of GRK5 (residues 563–590) contains residues autophosphorylated in the presence of calmodulin as well as the residues phosphorylated by protein kinase C. Deletion of this domain increases the apparent affinity of GRK5 for receptor substrates 3–4-fold but has no effect on nonreceptor substrates. These findings define residues 563–590 of GRK5 as an autoinhibitory domain with efficacy that is regulated by phosphorylation. Another C-terminal domain in GRK5 that appears to be functionally important is found between residues 552 and 562. Deletion of this region significantly inhibits kinase phosphorylation of membrane-bound receptor substrates but has no effect on soluble substrates. Additional studies reveal that this domain is critical for GRK5 interaction with phospholipids and for the intracellular localization of the kinase. Interestingly, similar regions in GRK4 and GRK6 appear to be palmitoylated (and involved in membrane interaction), suggesting evolutionary conservation of the function of this domain. Many G protein-coupled receptors are phosphorylated and regulated by a distinct family of G protein-coupled receptor kinases (GRKs) that specifically target the activated form of the receptor. Recent studies have revealed that the GRKs are also subject to post-translational regulation. For example, GRK5 activity is strongly inhibited by protein kinase C phosphorylation and by Ca2+-calmodulin binding. Ca2+-calmodulin binding also promotes GRK5 autophosphorylation, which further contributes to kinase inhibition. In this study we identify two important structural domains in GRK5, a phospholipid binding domain (residues 552–562) and an autoinhibitory domain (residues 563–590), that significantly contribute to GRK5 localization and function. We demonstrate that the C-terminal region of GRK5 (residues 563–590) contains residues autophosphorylated in the presence of calmodulin as well as the residues phosphorylated by protein kinase C. Deletion of this domain increases the apparent affinity of GRK5 for receptor substrates 3–4-fold but has no effect on nonreceptor substrates. These findings define residues 563–590 of GRK5 as an autoinhibitory domain with efficacy that is regulated by phosphorylation. Another C-terminal domain in GRK5 that appears to be functionally important is found between residues 552 and 562. Deletion of this region significantly inhibits kinase phosphorylation of membrane-bound receptor substrates but has no effect on soluble substrates. Additional studies reveal that this domain is critical for GRK5 interaction with phospholipids and for the intracellular localization of the kinase. Interestingly, similar regions in GRK4 and GRK6 appear to be palmitoylated (and involved in membrane interaction), suggesting evolutionary conservation of the function of this domain. G protein-coupled receptor green fluorescent protein glutathione S-transferase G protein-coupled receptor kinase phosphate-buffered saline phosphatidylcholine polymerase chain reaction phosphatidylinositol-4,5-bisphosphate protein kinase C rod outer segments. A large variety of extracellular stimuli transmit their signals via interaction with cell surface G protein-coupled receptors (GPCRs)1 (1Strader C.D. Fong T.M. Graziano M.P. Tota M.R. FASEB J. 1995; 9: 745-754Crossref PubMed Scopus (327) Google Scholar). A fundamental process of regulating the responsiveness of a system, termed desensitization, uses numerous mechanisms initiated by phosphorylation of the receptor. Receptor phosphorylation results in uncoupling of receptor and G protein and may also control processes such as GPCR internalization and down-regulation. The phosphorylation of GPCRs can be mediated either by second messenger-dependent kinases, such as protein kinase A and protein kinase C (PKC), or by G protein-coupled receptor kinases (GRKs). The six mammalian GRKs identified to date can be divided into three subfamilies based on their sequence homology (2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (853) Google Scholar, 3Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (468) Google Scholar): GRK1 (rhodopsin kinase) forms one group; GRK2 (β-adrenergic receptor kinase) and GRK3 constitute a second; and GRK4, GRK5, and GRK6 constitute the third subfamily. One feature of GRKs that plays a critical role in regulating the interaction of the soluble kinases with integral membrane GPCRs is their ability to associate with the plasma membrane. Interestingly, the various GRKs use distinct mechanisms for membrane interaction, including modification of the C-terminal domain by lipids (GRK1, GRK4 and GRK6) and association with Gβγ subunits and acidic phospholipids (GRK2 and GRK3) (4Stoffel III, R.H. Pitcher J.R. Lefkowitz R.J. J. Membr. Biol. 1997; 157: 1-8Crossref PubMed Scopus (43) Google Scholar). The specific determinants of membrane interaction for GRK5 are poorly understood. Although GRK5 does not appear to be modified by lipids, it displays a high level of membrane association, possibly via direct binding of a C-terminal polybasic domain with phospholipids (5Kunapuli P. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1994; 269: 10209-10212Abstract Full Text PDF PubMed Google Scholar). However, it has been also suggested that GRK5 contains an N-terminal phosphatidylinositol-4,5-bisphosphate (PIP2) binding site that can promote membrane association (6Pitcher J.A. Fredericks Z.L. Stone W.C. Premont R.T. Stoffel R.H. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 24907-24913Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Additional mechanisms of GRK regulation have been identified. Of particular interest is the calcium-dependent regulation of GRK activity. Recent studies have demonstrated that GRK2 can be phosphorylated by PKC, resulting in an ∼2–3-fold activation of the kinase, possibly via an increased association of GRK2 with membranes (7Chuang 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, 8Winstel R. Freund S. Krasel C. Hoppe E. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2105-2109Crossref PubMed Scopus (141) Google Scholar). Conversely, GRK5 is significantly inhibited after phosphorylation by PKC because of a decreased activity and decreased affinity for receptor (9Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In the visual system, GRK1 has been shown to be inhibited by the Ca2+-binding protein recoverin (10Chen 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). Although regulation by recoverin may be specific for GRK1, a universal mediator of calcium signaling, calmodulin, appears to inhibit all other GRK subtypes, with GRK5 being the most sensitive (11Chuang T.T. Paolucci L. De Blasi A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, H. 1997; PubMed Scopus Google Scholar, A.N. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus (135) Google Scholar). We have demonstrated that calmodulin of GRK5 and that this phosphorylation inhibits GRK5 in a similar to the phosphorylation A.N. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus (135) Google Scholar). we that the residues autophosphorylated in the presence of calmodulin well as the residues phosphorylated by a C-terminal region of GRK5 (residues region of GRK5 appears to function as an autoinhibitory because increases GRK5 affinity for phospholipids and receptor. We also demonstrate that the sequence 552–562) of this region is critical for GRK5 association with phospholipids and for the intracellular localization of the kinase. studies reveal two structural domains that an important role in GRK5 function and regulation. the high polymerase chain reaction system, and other or phosphatidylcholine contains GRK5 (residues and GRK6 (residues and to GRK5 residues and by the on an and by high other (9Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). for GRKs by the sequence of GRK5 P. Benovic J.L. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google in the GRK5 and the of the in their the chain to in and with of the after and GRKs by on as A.N. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus (135) Google Scholar). GRK of of the GRK5 by with or calmodulin in of for by on and of A and of (9Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). on for after which of a protein and the an The with of and with two of to and of on a PubMed Scopus Google Scholar). The and and the and The activity of GRK5 in and by with either various of rod outer membranes or in of The of calmodulin in for in with and on a with and to and to the of for the of GRK5 in a of the GRK5 to and by with to the level of GRK5 of by of and GRK5 by In studies the effect of on phosphorylation of and GRK5 and in the presence or of with the and as membranes as H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). by of phosphatidylcholine with or of either or in of on for The ability of GRK5 to associate with phospholipids and by of GRK5 in and in the presence or of of phospholipid or membranes of in of on for The of in The for and the in of reaction of the and to and by with of by and the of GRK5 as a of the various regions of GRK5 the and to in the The of the in their the chain The in and The of the as by by binding as a the ability of to of the protein in the presence or of phosphatidylcholine in of on for and by as by to with of or to the and in a after of the The with phosphate-buffered saline after and with for three with and with for These for with and and with in the for three with and for The with a in and for with and for by with for with and on a with of green fluorescent protein or receptor to that with the that and not in the on a a GRK5 and as (5Kunapuli P. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1994; 269: 10209-10212Abstract Full Text PDF PubMed Google Scholar). GRK5 via of GRK5 in of and either phosphatidylcholine or for phosphorylation GRK5 by on as (9Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). of the GRK by this calmodulin not to of GRK5 of with of A with or of The for on and The to of saline with of either GRK5 residues or GRK6 residues on for of a protein and the an The with of The of by the studies have the C-terminal region of GRKs in membrane association (4Stoffel III, R.H. Pitcher J.R. Lefkowitz R.J. J. Membr. Biol. 1997; 157: 1-8Crossref PubMed Scopus (43) Google Scholar). Although and phospholipid binding domains have been identified in most of the specific regions of GRK5 involved in membrane interaction have not been A polybasic domain the C of GRK5 has been to a role in phospholipid association (5Kunapuli P. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1994; 269: 10209-10212Abstract Full Text PDF PubMed Google Scholar). Interestingly, the C-terminal region of GRK5 has also been identified as the site of phosphorylation by protein kinase a modification that results in of GRK5 activity (9Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). further define the role of the C-terminal region of GRK5 in phospholipid binding and interaction, we a of C-terminal of GRK5 with or residues the C in and activity of residues in poorly that not Interestingly, the various a effect on the ability of GRK5 to substrates. of the soluble protein by of to residues of GRK5 A and In phosphorylation of on the of the C-terminal sequence phosphorylation increased residues the C and the of or residues and in of phosphorylation A and A of phosphorylation revealed that of C-terminal residues increased the affinity of GRK5 for decreased for GRK5 to for and no effect on of residues in no in and an in The of residues an in affinity for of and with The increased affinity of the GRK5 C-terminal for also we their ability to to in which of GRK5 with of and not In of residues a in kinase binding to to of the C-terminal domain of GRK5 a effect on phosphorylation of the membrane-bound but no effect on phosphorylation of the soluble we the various GRK5 interaction with We and have shown that GRK5 can associate and with phospholipid (5Kunapuli P. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1994; 269: 10209-10212Abstract Full Text PDF PubMed Google Scholar, A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (110) Google R.T. Koch W.J. Inglese J. Lefkowitz R.J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). In we a that contains as well as numerous other We found that the of GRK5 that on the of phospholipid in the we to for GRK5 binding by the phospholipid The binding of GRK5 not and an affinity and The of residues in an of phospholipid binding with an of phospholipid of residues no effect not studies have suggested an important role of an N-terminal binding domain in GRK5 interaction with J.A. Fredericks Z.L. Stone W.C. Premont R.T. Stoffel R.H. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 24907-24913Abstract Full Text Full Text PDF PubMed Scopus (135) Google we the of on GRK5 binding to The of to the the affinity of GRK5 and 3–4-fold and of in an in the affinity of a binding site in GRK5 of the C-terminal phospholipid binding domain identified in this The interaction of the C-terminal residues of the kinase with phospholipids further demonstrated A to GRK5 residues for interaction with phospholipids and inhibited GRK5 binding to with an of A and A a of the phospholipid binding site (residues no effect on GRK5 binding to phospholipids to with the ability of to inhibit GRK5 membrane association, this inhibited the phosphorylation of no effect on the kinase activity A of phosphorylation revealed that the presence of decreased the affinity of GRK5 for and the not of a effect on phosphorylation The ability of residues of GRK5 to promote interaction with phospholipids also shown a of A protein residues of GRK5 no ability to phospholipid the of residues in phospholipid binding protein The binding of GRK5 to phospholipid appears to be to because significantly inhibit phospholipid binding of GRK5 and not the role of the C-terminal phospholipid binding domain in the intracellular localization of GRK5, GRK5 in and by The of GRK5 is with a of the kinase being with the plasma membrane and the of GRK5 appears similar to that of an receptor a protein that is the plasma membrane. a to that of GRK5 C and the ability of this protein to associate with the phospholipid In the of a with a localization and the of similar to that of a soluble protein that to the and These results demonstrate that the C-terminal phospholipid binding domain of GRK5 (residues 552–562) plays a critical role in the intracellular localization of this kinase. studies have demonstrated that PKC phosphorylation of GRK5, residues results in of GRK5 activity (9Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). also significantly inhibits GRK5 activity A.N. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus (135) Google we the to the site autophosphorylated in the presence of calmodulin also the C-terminal domain of Although the of C-terminal residues no effect on autophosphorylation, the of residues that residues autophosphorylated in the presence of calmodulin of region and and is distinct the phosphorylated by that the site is the C of GRK5, we an residues that is of the kinase. GRK5 via in the presence of either phospholipids or divided in with or for with and to in after and the C-terminal by the significantly the the reaction with GRK5 autophosphorylated in the presence of phospholipids The phosphorylated to be and (5Kunapuli P. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1994; 269: 10209-10212Abstract Full Text PDF PubMed Google R.T. Koch W.J. Inglese J. Lefkowitz R.J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). In no on the the reaction with GRK5 autophosphorylated in the presence of calmodulin that the the sequence we that GRK5 has a calmodulin binding domain the of the protein (residues A.N. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus (135) Google Scholar). We identified a C-terminal site GRK5 that also with calmodulin (residues A.N. Benovic J.L. 1998; Scopus Google Scholar). However, a the N-terminal calmodulin binding which a in affinity of this site for calmodulin, no effect on calmodulin of phosphorylation by GRK5 A.N. Benovic J.L. 1998; Scopus Google Scholar). the no effect on of GRK5 that calmodulin interaction with the C-terminal binding domain on GRK5 plays the role in GRK5 further define the role of the C-terminal calmodulin binding domain of GRK5, we the effect of calmodulin on the various C-terminal of inhibited phosphorylation by GRK5 with an of C and and inhibited in a similar to that of the kinase. In the of residues an in calmodulin of GRK5 the of residues no These results that the C-terminal calmodulin binding domain GRK5 (residues 552–562) with the phospholipid binding domain on GRKs an important role in regulating numerous G protein-coupled the activity of GRKs is also Although mechanisms appear GRK subtypes, are GRKs are a their to the plasma membrane GPCRs GRKs use distinct mechanisms to their membrane association (4Stoffel III, R.H. Pitcher J.R. Lefkowitz R.J. J. Membr. Biol. 1997; 157: 1-8Crossref PubMed Scopus (43) Google Scholar). GRK1 a C-terminal that and of the kinase PubMed Scopus Google Scholar, J. Lefkowitz R.J. J. Biol. Chem. Full Text PDF PubMed Google which are critical for GRK1 membrane association and activity J. Lefkowitz R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Koch W.J. Lefkowitz R.J. PubMed Scopus Google Scholar). GRK2 and GRK3 a C-terminal homology domain that association of kinases with Gβγ subunits and acidic phospholipids PubMed Scopus Google Scholar, J.A. Inglese J. J.B. J.L. C. Benovic J.L. Lefkowitz R.J. PubMed Scopus Google Scholar, Benovic J.L. Google Scholar, J. E. Benovic J.L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. Lefkowitz R.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, Pitcher J.A. Stoffel R.H. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). GRK4 and GRK6 are palmitoylated C-terminal residues R.T. Stoffel R.H. Pitcher J.A. C. Inglese J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, R.H. Premont R.T. Lefkowitz R.J. Inglese J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). of the in GRK6 results in a affinity for membranes and receptor phosphorylation Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Although no have been identified for GRK5, the protein with phospholipid The C-terminal polybasic domain has been in phospholipid binding (5Kunapuli P. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1994; 269: 10209-10212Abstract Full Text PDF PubMed Google Scholar). However, of residues this domain has no apparent effect on receptor phosphorylation (6Pitcher J.A. Fredericks Z.L. Stone W.C. Premont R.T. Stoffel R.H. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 24907-24913Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). In this study we a of the role of the C-terminal domain of GRK5 in receptor phosphorylation and association with phospholipid The of C-terminal residues of GRK5 no direct effect on the activity of the kinase, as phosphorylation of a soluble such as However, this an in the affinity of GRK5 for The phospholipid binding of this region of GRK5 appear to be to an region of the kinase (residues of this domain to a to promote phospholipid binding of the resulting and the this sequence inhibited kinase interaction with These that residues constitute a phospholipid binding domain of GRK5 critical for interaction with The binding of GRK5 to phospholipids an interaction between phospholipid and the residues of GRK5 (5Kunapuli P. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1994; 269: 10209-10212Abstract Full Text PDF PubMed Google Scholar, J.A. Fredericks Z.L. Stone W.C. Premont R.T. Stoffel R.H. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 24907-24913Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). study that significantly inhibits GRK5 binding to further the of this The C-terminal phospholipid binding domain of GRK5 contains residues and with and also possibly involved in phospholipid A that this region of GRK5 be involved in interaction with (4Stoffel III, R.H. Pitcher J.R. Lefkowitz R.J. J. Membr. Biol. 1997; 157: 1-8Crossref PubMed Scopus (43) Google Scholar). However, the of to the phospholipid in no effect on GRK5 binding not demonstrate that residues of GRK5 constitute a phospholipid binding the of the lipids with this domain to be Interestingly, similar regions of GRK4 (residues and GRK6 (residues a level of homology with the phospholipid binding domain of GRK5, also residues that appear to be palmitoylated In the C-terminal region of GRK1 homology with the other it a for the C-terminal regions of GRK1, and appear involved in phospholipid One that this region as a direct phospholipid binding domain that mechanisms for membrane and is also by the of the which an between and that is in the for GRK4 R.T. Stoffel R.H. Pitcher J.A. C. Inglese J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google GRK5, and GRK6 and J. L. The residues of the C-terminal phospholipid binding domain the residues palmitoylated in GRK4 and GRK6 has been demonstrated that GRK5 interaction with may residues (6Pitcher J.A. Fredericks Z.L. Stone W.C. Premont R.T. Stoffel R.H. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 24907-24913Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). that GRK5 binding to because the of to phospholipids results in an in the affinity of GRK5 for phospholipid has an effect on the C-terminal binding affinity for is that the binding of to phospholipids is to the presence of of in the However, the of the site to be because significantly association with with and this association not by the presence of not The C-terminal phospholipid binding domain also appears to be critical for the intracellular localization of GRK5 GRK5 in displays a high of localization the plasma suggesting that GRK5 is membrane in In the of residues results in a of the kinase. These that interaction with is not for GRK5 binding to the and that the C-terminal binding domain is critical for membrane interaction be involved in GRK5 to specific regions of the cell with high of this the C-terminal phospholipid binding domain also appears important for the function of Although of residues no effect on phosphorylation of the soluble protein it in a for phosphorylation of membrane-bound The of for phosphorylation that interaction of the C-terminal domain with phospholipids not kinase association with membrane but also increases the activity of GRK5 for GPCR phosphorylation. Interestingly, the of residues a significantly effect on the affinity of GRK5 for phospholipid with with a affinity for and an affinity for the of phospholipid interaction by the in an in the affinity of GRK5 for that other regions of GRK5 contribute to kinase binding to most via interaction with and is with findings GRKs can with of GPCRs in the of lipids J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, Benovic J.L. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Recent that GRK activity is regulated by GRK1 is specifically inhibited by the Ca2+-binding protein recoverin (10Chen 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 the other GRKs can be inhibited by calmodulin (11Chuang T.T. Paolucci L. De Blasi A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, H. 1997; PubMed Scopus Google Scholar, A.N. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus (135) Google Scholar). to GRK5 with high affinity and inhibits GRK5 binding to membranes and phospholipid and GRK5 phosphorylation of (11Chuang T.T. Paolucci L. De Blasi A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, A.N. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus (135) Google Scholar). appears to to two distinct domains on GRK5, one in the (residues A.N. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus (135) Google and one in the C (residues A.N. Benovic J.L. 1998; Scopus Google Scholar). In the study we demonstrate that is as sensitive to calmodulin as is and GRK5, has to These results that the C-terminal calmodulin binding site residues the domain involved in phospholipid binding. calmodulin binding to the C-terminal site be to inhibit GRK5 interaction with phospholipid phosphorylation of receptor substrates. of calmodulin and phospholipid binding domains may as a for regulation of such as J. J. Biol. Chem. Full Text PDF PubMed Google H. S. J. Biol. Chem. Full Text PDF PubMed Google and H. 1996; PubMed Scopus Google have calmodulin binding that are involved in phospholipid The other in which calmodulin GRK5 activity is via kinase A.N. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus (135) Google Scholar). inhibits GRK5 phosphorylation of and binding to The autophosphorylated in the presence of calmodulin the sequence of GRK5, of the domain (residues binding may a in the C-terminal domain of GRK5, it to the The sequence is also to the region phosphorylated by PKC The of two may and PKC phosphorylation have on GRK5 activity. on we can the role of the C-terminal domain (residues 563–590) in GRK5 regulation that this sequence is involved in a direct interaction with either phospholipids or because of this domain has no effect on the kinase. In of this region results in a 3–4-fold increased affinity of GRK5 for phospholipids and we that this region of GRK5 may function as an autoinhibitory domain to inhibit the activity of Deletion of residues is to GRK5 interaction with phospholipids and The region (residues to as an of autoinhibitory function. calcium the cell this results in increased phosphorylation of GRK5 either activation of PKC or via The resulting phosphorylated residues be to with the C-terminal into an of the GRK4 that GRK5 has a that is not in GRK4 or GRK6 the presence of this autoinhibitory domain may be to GRK4 and GRK6 are a modification that has been demonstrated to be important for GRK6 function Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). is a one that promote GRK4 and GRK6 the kinases GRK5 appears to with phospholipids via residues and displays an apparent membrane association, it other of regulation. is that the autoinhibitory domain has to phosphorylation of receptors and to a to GRK5 in a the mechanisms that to of GRK5 activation of receptors to In studies two important structural domains in the C of GRK5 that significantly contribute to kinase function. form a phospholipid binding which with a calmodulin binding and is important for the intracellular localization of the 563–590 constitute an autoinhibitory the efficacy of which is by PKC phosphorylation or We R. for and of the