The Structure of G Protein-coupled Receptor Kinase (GRK)-6 Defines a Second Lineage of GRKs

We describe the 2.6-Å crystal structure of human G protein-coupled receptor kinase (GRK)-6, a key regulator of dopaminergic signaling and lymphocyte chemotaxis. GRK6 is a member of the GRK4 subfamily of GRKs, which is represented in most, if not all, metazoans. Comparison of GRK6 with GRK2 confirms that the catalytic core of all GRKs consists of intimately associated kinase and regulator of G protein signaling (RGS) homology domains. Despite being in complex with an ATP analog, the kinase domain of GRK6 remains in an open, presumably inactive conformation, suggesting that G protein-coupled receptors activate GRKs by inducing kinase domain closure. The structure reveals a putative phospholipid-binding site near the N terminus of GRK6 and structural elements within the kinase substrate channel that likely influence G protein-coupled receptor access and specificity. The crystalline GRK6 RGS homology domain forms an extensive dimer interface using conserved hydrophobic residues distinct from those in GRK2 that bind Gαq, although dimerization does not appear to occur in solution and is not required for receptor phosphorylation. We describe the 2.6-Å crystal structure of human G protein-coupled receptor kinase (GRK)-6, a key regulator of dopaminergic signaling and lymphocyte chemotaxis. GRK6 is a member of the GRK4 subfamily of GRKs, which is represented in most, if not all, metazoans. Comparison of GRK6 with GRK2 confirms that the catalytic core of all GRKs consists of intimately associated kinase and regulator of G protein signaling (RGS) homology domains. Despite being in complex with an ATP analog, the kinase domain of GRK6 remains in an open, presumably inactive conformation, suggesting that G protein-coupled receptors activate GRKs by inducing kinase domain closure. The structure reveals a putative phospholipid-binding site near the N terminus of GRK6 and structural elements within the kinase substrate channel that likely influence G protein-coupled receptor access and specificity. The crystalline GRK6 RGS homology domain forms an extensive dimer interface using conserved hydrophobic residues distinct from those in GRK2 that bind Gαq, although dimerization does not appear to occur in solution and is not required for receptor phosphorylation. G protein-coupled receptor kinases (GRKs) 2The abbreviations used are: GRKs, G protein-coupled receptor kinases; GPCRs, G protein-coupled receptors; RGS, regulator of G protein signaling; RH, RGS homology; PKA, protein kinase A; PKB, protein kinase B; PH, pleckstrin homology; AMPPNP, 5′-adenylyl β,γ-imidodiphosphate; IP3, inositol 1,4,5-trisphosphate; GSK3β, glycogen synthase kinase-3β. initiate homologous desensitization by phosphorylating the third cytoplasmic loop or tail of activated G protein-coupled receptors (GPCRs) (1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar, 2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (858) Google Scholar), thereby allowing cells to adapt to changing extracellular signals. The seven mammalian GRKs are grouped into three subfamilies based on sequence identity and gene structure (3Premont R.T. Macrae A.D. Aparicio S.A. Kendall H.E. Welch J.E. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 29381-29389Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The GRK1 subfamily consists of rhodopsin kinase (GRK1) and GRK7; the GRK2 subfamily consists of β-adrenergic receptor kinase-1 and -2 (GRK2 and GRK3); and the GRK4 subfamily consists of GRK4–6. All GRKs have an ∼500-amino acid structural core consisting of a regulator of G protein signaling (RGS) homology (RH) domain (4Siderovski D.P. Hessel A. Chung S. Mak T.W. Tyers M. Curr. Biol. 1996; 6: 211-212Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) and a kinase domain closely related to those of other AGC family members (5Benovic J.L. De Blasi A. Stone W.C. Caron M.G. Lefkowitz R.J. Science. 1989; 246: 235-240Crossref PubMed Scopus (331) Google Scholar), including protein kinase A (PKA) and protein kinase B (PKB). However, each GRK subfamily has distinct N and C termini containing elements known to regulate kinase activity and to mediate membrane targeting (1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar, 6Krasel C. Dammeier S. Winstel R. Brockmann J. Mischak H. Lohse M.J. J. Biol. Chem. 2001; 276: 1911-1915Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 7Pitcher J.A. Tesmer J.J. Freeman J.L. Capel W.D. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 34531-34534Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 8Noble B. Kallal L.A. Pausch M.H. Benovic J.L. J. Biol. Chem. 2003; 278: 47466-47476Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The GRK2 and GRK4 subfamilies diverged >1 billion years ago and are present in most, if not all, metazoans, including Drosophila melanogaster and Caenorhabditis elegans (3Premont R.T. Macrae A.D. Aparicio S.A. Kendall H.E. Welch J.E. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 29381-29389Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The GRK1 subfamily is thus far found only in vertebrates. To date, the defining structural features of the GRK family have been surmised primarily through crystal structures of only one member, bovine GRK2. In the crystal structures of GRK2 (9Lodowski D.T. Barnhill J.F. Pyskadlo R.M. Ghirlando R. Sterne-Marr R. Tesmer J.J. Biochemistry. 2005; 44: 6958-6970Crossref PubMed Scopus (47) Google Scholar) and its complexes with Gβγ (10Lodowski D.T. Pitcher J.A. Capel W.D. Lefkowitz R.J. Tesmer J.J. Science. 2003; 300: 1256-1262Crossref PubMed Scopus (318) Google Scholar) and Gαq (11Tesmer V.M. Kawano T. Shankaranarayanan A. Kozasa T. Tesmer J.J. 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The first binds the C-terminal pleckstrin homology (PH) domain of GRK2, by which the enzyme is recruited to the membrane via binding activated Gβγ subunits (16Haga K. Haga T. J. Biol. Chem. 1992; 267: 2222-2227Abstract Full Text PDF PubMed Google Scholar, 17Pitcher 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 (573) Google Scholar, 18Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar), and the second binds activated Gαq subunits (19Carman C.V. Parent J.L. Day P.W. Pronin A.N. Sternweis P.M. Wedegaertner P.B. Gilman A.G. Benovic J.L. Kozasa T. J. Biol. Chem. 1999; 274: 34483-34492Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 20Sallese M. Mariggio S. D'Urbano E. Iacovelli L. De Blasi A. Mol. Pharmacol. 2000; 57: 826-831Crossref PubMed Scopus (122) Google Scholar, 21Usui H. 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Chem. 1999; 274: 34483-34492Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). the GRK kinase domain is conserved the subfamilies sequence the RH domain is and the GRK N and C termini have or sequence the structure and of the domain the kinase domain conformation, and the membrane in other GRK subfamilies is not the RH domains of GRK1 and GRK4 subfamily members a in To the 2.6-Å crystal structure of the member of the GRK4 subfamily (3Premont R.T. Macrae A.D. Aparicio S.A. Kendall H.E. Welch J.E. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 29381-29389Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, J.L. J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, R.P. B. Benovic J.L. 1996; PubMed Google Scholar, A. M. M. S. C. J. 1999; PubMed Scopus Google Scholar). of GRK6 and receptor signaling Pharmacol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, R.T. Lefkowitz R.J. Caron M.G. Annu. Rev. 2004; PubMed Scopus Google Scholar, M. 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Biol. 2003; PubMed Scopus Google Scholar), although GRK6 from the to for GRK2. on a with and GRK6 to in a and in The of GRK6 from to of of the GRK6 protein a of for that GRK6 is via in is GRK2 J.A. Tesmer J.J. Freeman J.L. Capel W.D. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 34531-34534Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). by in the of and The of required for The of of protein and of solution containing or acid and in and the of of with of GRK6 to the with a and and and two in the and by with an and by into a solution consisting of acid AMPPNP, and The through a of to and in from on an To to the which to the of the crystal to the of the crystal by in the in two using or and or for a of the crystal in the to The and using J. T. S. A. 2004; PubMed Scopus Google Scholar) and associated from J. 2003; PubMed Scopus Google Scholar) and by by the R.J. Biol. 2004; PubMed Scopus Google Scholar), with the RH and kinase domains of GRK2 from with inositol or glycogen synthase J. V.M. Thompson Biol. PubMed Scopus Google Scholar). However, not for for two of and in J. M. R.J. T. Biol. 1998; PubMed Scopus Google Scholar) to The GRK6 using and in J. 2003; PubMed Scopus Google Scholar). each of the into using the M. A. PubMed Scopus Google Scholar). The of the using J. 1993; Google Scholar). and with The Scholar) and S. M.J. J.A. S. A. 2001; PubMed Scopus Google Scholar). of the RH the GRK6 a of human GRK6 J.L. J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar) with and into and and and in using the and the of the for GRK6 and its from cells and in A.N. R.P. Benovic J.L. PubMed Scopus Google Scholar). protein by with a GRK6 and its in containing in the not rhodopsin phosphorylation. and using a and with with of GRK6 three to and in and and in an three and in a using by the a with a of to a of and a of that GRK6 of is each a with a The crystal structure of the human complex using to 2.6-Å with by of GRK6 A and are present in the of the and a that of The residues and in A and residues and in B of the protein used for that the are and not The residues from to in each to the of the kinase domain S. S.S. 1997; 5: Full Text Full Text PDF PubMed Scopus Google Scholar), which in structures of GRK2. A is to residues near the N terminus of the protein which of an phospholipid-binding site in each J.A. Stone W.C. R.T. W.J. Lefkowitz R.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). to the in each and residues of each GRK6 to its to the first in each However, to residues from the C terminus of each which are and and and in to the of to is the of to in to the of to used of the from to in to the of to is the of in to the of to used of the from to in a The of sequence GRKs is the kinase which residues in in other protein the catalytic site of the GRK6 kinase domain is in a a and and a Despite the of in the site the kinase domain of GRK6 in an inactive conformation with a is for an AGC the binding of with kinase domain and of the (15Taylor S.S. Yang J. Wu J. Haste N.M. Radzio-Andzelm E. Anand G. Biochim. Biophys. Acta. 2004; 1697: 259-269Crossref PubMed Scopus (260) Google Scholar). the of GRK6 and GRK2, are with to each However, a conformation an to the conformation of J. V.M. Thompson Biol. PubMed Scopus Google Scholar). In the of required to the conformation is to the of the kinase to GRK6 (GRK2 within the The structure of in the GRK6 site of a GRK catalytic site with other AGC kinases J. V.M. Thompson Biol. PubMed Scopus Google Scholar, H. R. J. 1993; PubMed Scopus Google Scholar). in PKA or PKB, the catalytic with the and of the ATP analog, and the into a hydrophobic for one of the two is in residues by the and of which are to the open conformation of the kinase the by GRKs is that of PKA or A and the of the tail and associated is by the to that of PKA or and has the conformation does in the structures of GRK2. for a in loop binding is in PKA and other protein kinases (15Taylor S.S. Yang J. Wu J. Haste N.M. Radzio-Andzelm E. Anand G. Biochim. Biophys. Acta. 2004; 1697: 259-269Crossref PubMed Scopus (260) Google Scholar, M. Kuriyan J. Full Text Full Text PDF PubMed Scopus Google Scholar). The structural for is not although that to within the The and of the GRK6 to in other AGC kinases that the of the In are and in GRK2 and In the GRK6 A of the loop the of the channel the N terminus of bind to PKA and In in the B are from the that the A is the B and that the A loop is by a crystal is that influence the structure of the However, the of residues and in the B that residues the GRK6 binds in a that is not to binds the J. V.M. Thompson Biol. PubMed Scopus Google Scholar), or the loop adopts an conformation substrate that residues are the channel of GRK6 to only with those of PKA and B and In the channel of GRK2 is and with residues to the site J.J. K. Caron M.G. Lefkowitz R.J. Benovic J.L. Biochemistry. PubMed Scopus Google Scholar). to a sequence in and GRK6 M.G. Pitcher J.A. Mol. Biol. 2004; PubMed Scopus Google Scholar). The of the GRK6 RH domain consists of associated into and and and of the GRK2 and GRK6 RH domains of for that are with to each other and thus are distinct from RH domains of the RGS protein family the two are to those of The GRK2 and GRK6 RH domains are N In GRK2, forms an interface with its In an additional is found the N terminus which additional the RH and kinase domains and a of residues in binding and and J.A. Stone W.C. R.T. W.J. Lefkowitz R.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A.N. Benovic J.L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). in the GRK2 and GRK4 the of the RH domain to have to with the that are of each The GRK6 RH domain does not have structural known to required for binding subunits in GRK2 or the RGS protein of GRK6 is two that of GRK2 and thereby residues to those for GRK2 binding to Gαq A and B; and R. Tesmer J.J. Day P.W. Stracquatanio R.P. Cilente J.A. O'Connor K.E. Pronin A.N. Benovic J.L. Wedegaertner P.B. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, P.W. C.V. Sterne-Marr R. Benovic J.L. Wedegaertner P.B. Biochemistry. 2003; PubMed Scopus Google Scholar). the a for binding or Gαq subunits in RGS Gilman A.G. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), has a distinct structure in that the GRK6 RH domain a protein for in a that to RGS However, the RH domain of GRK6 does a conserved extensive protein dimer interface using a similar to that used by the RH domain of to bind a and and W.D. Sternweis Mol. Biol. 2005; PubMed Scopus Google Scholar). The interface of and features a interaction the C termini of each GRK6 and an the of and and dimer and and to the core of the residues are conserved each of in GRK1 and are conserved in and GRK4 which into the site of W.D. Sternweis Mol. Biol. 2005; PubMed Scopus Google Scholar), from the RH domain from a that is to GRK6 However, is for a interaction GRK6 and subunits (19Carman C.V. Parent J.L. Day P.W. Pronin A.N. Sternweis P.M. Wedegaertner P.B. Gilman A.G. Benovic J.L. Kozasa T. J. Biol. Chem. 1999; 274: 34483-34492Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Despite the of the dimer interface and the of residues that GRK6 a in solution by and not However, dimerization occur the membrane the of GRK6 is or activated GPCRs, that are S. M. 2004; 5: PubMed Scopus Google Scholar, G. Mol. Pharmacol. 2004; PubMed Scopus Google Scholar). We the hydrophobic residues within the dimer interface are required for receptor phosphorylation. The and to dimer by The in and all rhodopsin to the GRK6 not not to a two of GRK6 and for to only receptor that dimerization of is that the residues are not required for the of activated of are in the The GRK6 RH and kinase domains are intimately associated through two that of and in GRK2. The is by the and from the RH the from the kinase The is the loop of the RH and the of the kinase domain interface is in GRK6 in GRK2 and a and hydrophobic that appear to conserved the GRK1 and GRK4 subfamilies The bipartite interaction the RH and kinase domains to conserved in all Despite in the domain core of GRK6 is in conformation that of GRK2. The binding of have in although structural the GRK2 and GRK4 of the kinase domain of GRK2 and the RH domain of GRK6 is with to that of GRK2, with the of through the of the and of is to that which the of the GRK2 and GRK6 kinase domains are of similar and the in the of the RH domain and the of GRK6 with to those of GRK2 appear to The of GRK6 with the membrane is in by the of residues that in a C-terminal that is not in crystal structure R.T. Lefkowitz R.J. Inglese J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In GRK4 subfamily members a putative site the N terminus a sequence that the RH and kinase domains in the structure of GRK6 J.A. Stone W.C. R.T. W.J. Lefkowitz R.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). All the first two residues of sequence are in crystal is in to residues and is to the or of or used The is via or with and the two residues being from the of the kinase residues are in the GRK4 subfamily The residues to a the of the to of GRK2 is by protein kinase C C. Dammeier S. Winstel R. Brockmann J. Mischak H. Lohse M.J. J. Biol. Chem. 2001; 276: 1911-1915Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), and of GRK1 is K. J. S.A. M.J. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). However, GRK2 is not to of residues and a GRK6 is not with and not have We that the of a binding site for an the or of Remarkably, the of the putative site and the N and C termini of which additional are in the with to the domain core the GRK2 and a and on RH and kinase domains that with the of sequence and structural the N and C termini of GRKs, the catalytic and are in the GRK4 and GRK2 likely that all GRKs with in a similar with kinase in to the membrane to receptor GRK2 is an for of W.J. Mol. Biochem. 2004; PubMed Scopus Google Scholar), GRK6 is a for of and M. A. Macrae A.D. Kim Lefkowitz R.J. Caron M.G. R.T. 2003; 38: Full Text Full Text PDF PubMed Scopus Google Scholar, E. L. Benovic J.L. 2005; PubMed Scopus Google Scholar). Comparison of the GRK2 and GRK6 structures reveals features of kinase domains that to the of for GRKs other AGC binding does not domain in GRK2 or GRK6 and the GRK to in a conformation that of PKA or PKB, the GRK a for with other AGC are the GRK6 and GRK2 kinase domains. have in with that GRK4 subfamily members and GRK2 J.J. K. Caron M.G. Lefkowitz R.J. Benovic J.L. Biochemistry. PubMed Scopus Google Scholar, J.J. M.M. Benovic J.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The loop one of the channel in GRK6 is in the GRK4 and GRK1 loop kinase activity an activated is bind to GRK6 in a from the complexes of PKA and The domain of GRK2 and GRK6 have structural not only within RH and kinase in domains are with to each Despite a similar bipartite interaction is the RH and kinase domains in an conserved for the to the kinase in its in the interface in the the C terminus of the kinase In and protein kinase with the only which is required for kinase activity in Biochem. J. Scopus Google Scholar, M. M. S. R.M. J. PubMed Scopus Google Scholar). GRK kinase domains have thus far to in a state similar to PKB, of the domain interface to the inactive conformation of the kinase similar to homology domain the kinase in Src family kinases (12Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1252) Google Scholar, 14Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1047) Google Scholar). of the interface required for kinase domain and the RH with the does the A of PKA, which the of its kinase domain (15Taylor S.S. Yang J. Wu J. Haste N.M. Radzio-Andzelm E. Anand G. Biochim. Biophys. Acta. 2004; 1697: 259-269Crossref PubMed Scopus (260) Google Scholar). the RH domain with the in GRK2 and of the domain interface to the of elements with to the kinase the of the in GRK2 and GRK6 are similar structural GRK6 a dimer using a of its RH domain that is conserved in the GRK4 and GRK1 subfamilies and GRK6 is a in dimerization from the protein within the GRK6 However, the and of the used for dimerization a the crystalline GRK6 dimer is not with GRK2 with remains that GRK1 and GRK4 family members with or receptor the dimer in a GRK2 using the in is that a dimer of GRK6 is required for other receptor M.G. Pitcher J.A. Mol. Biol. 2004; PubMed Scopus Google Scholar) or that the interface used for dimerization in the crystal is used to bind of protein in The GRK kinase domain has been to an open, presumably inactive conformation in crystal one in the structure (10Lodowski D.T. Pitcher J.A. Capel W.D. Lefkowitz R.J. Tesmer J.J. Science. 2003; 300: 1256-1262Crossref PubMed Scopus (318) Google Scholar), in the GRK2 structure (9Lodowski D.T. Barnhill J.F. Pyskadlo R.M. Ghirlando R. Sterne-Marr R. Tesmer J.J. Biochemistry. 2005; 44: 6958-6970Crossref PubMed Scopus (47) Google Scholar), one in the structure (11Tesmer V.M. Kawano T. Shankaranarayanan A. Kozasa T. Tesmer J.J. Science. 2005; 310: 1686-1690Crossref PubMed Scopus (246) Google Scholar), and two in the open conformation is a a the structure that the binding of does not to kinase domain closure. of the GRK kinase domain is not required for is in other AGC kinases Biochem. J. Scopus Google Scholar), all for appear to in GRK structures for kinase domain closure. We that receptor binding to which or to the of the and the N terminus of the enzyme first elements are not in the GRK2 and GRK6 crystal The is to within the and with does in The GRK N terminus is required for receptor and thereby in receptor or binding or in the structural from the inactive to state of the kinase B. Kallal L.A. Pausch M.H. Benovic J.L. J. Biol. Chem. 2003; 278: 47466-47476Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, L. G. J. 1999; PubMed Scopus Google Scholar, K. J. L. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). of GRKs in an conformation additional on the of structural elements and the for of GRKs by We J. T. and other members of the for and that of GRK6 We T. and for and R. Sterne-Marr for rhodopsin used in of the with