title: Structure and Function of the Hypertension Variant A486V of G Protein-coupled Receptor Kinase 4.
abstract: G-protein-coupled receptor (GPCR) kinases (GRKs) bind to and phosphorylate GPCRs, initiating the process of GPCR desensitization and internalization. GRK4 is implicated in the regulation of blood pressure, and three GRK4 polymorphisms (R65L, A142V, and A486V) are associated with hypertension. Here, we describe the 2.6 Å structure of human GRK4α A486V crystallized in the presence of 5'-adenylyl β,γ-imidodiphosphate. The structure of GRK4α is similar to other GRKs, although slight differences exist within the RGS homology (RH) bundle subdomain, substrate-binding site, and kinase C-tail. The RH bundle subdomain and kinase C-terminal lobe form a strikingly acidic surface, whereas the kinase N-terminal lobe and RH terminal subdomain surfaces are much more basic. In this respect, GRK4α is more similar to GRK2 than GRK6. A fully ordered kinase C-tail reveals interactions linking the C-tail with important determinants of kinase activity, including the αB helix, αD helix, and the P-loop. Autophosphorylation of wild-type GRK4α is required for full kinase activity, as indicated by a lag in phosphorylation of a peptide from the dopamine D1 receptor without ATP preincubation. In contrast, this lag is not observed in GRK4α A486V. Phosphopeptide mapping by mass spectrometry indicates an increased rate of autophosphorylation of a number of residues in GRK4α A486V relative to wild-type GRK4α, including Ser-485 in the kinase C-tail.
authors.affiliation: From Screening and Protein Sciences, Merck Research Laboratories, North Wales, Pennsylvania 19454 and samantha_allen@merck.com.
authors.forename: Samantha J
authors.initials: SJ
authors.lastname: Allen
authors.affiliation: Structural Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486.
authors.forename: Gopal
authors.initials: G
authors.lastname: Parthasarathy
authors.affiliation: From Screening and Protein Sciences, Merck Research Laboratories, North Wales, Pennsylvania 19454 and.
authors.forename: Paul L
authors.initials: PL
authors.lastname: Darke
authors.affiliation: From Screening and Protein Sciences, Merck Research Laboratories, North Wales, Pennsylvania 19454 and.
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Diehl
authors.affiliation: From Screening and Protein Sciences, Merck Research Laboratories, North Wales, Pennsylvania 19454 and.
authors.forename: Rachael E
authors.initials: RE
authors.lastname: Ford
authors.affiliation: From Screening and Protein Sciences, Merck Research Laboratories, North Wales, Pennsylvania 19454 and.
authors.forename: Dawn L
authors.initials: DL
authors.lastname: Hall
authors.affiliation: Structural Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Johnson
authors.affiliation: Structural Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486.
authors.forename: John C
authors.initials: JC
authors.lastname: Reid
authors.affiliation: From Screening and Protein Sciences, Merck Research Laboratories, North Wales, Pennsylvania 19454 and.
authors.forename: Keith W
authors.initials: KW
authors.lastname: Rickert
authors.affiliation: From Screening and Protein Sciences, Merck Research Laboratories, North Wales, Pennsylvania 19454 and.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Shipman
authors.affiliation: Structural Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Soisson
authors.affiliation: From Screening and Protein Sciences, Merck Research Laboratories, North Wales, Pennsylvania 19454 and.
authors.forename: Paul
authors.initials: P
authors.lastname: Zuck
authors.affiliation: From Screening and Protein Sciences, Merck Research Laboratories, North Wales, Pennsylvania 19454 and.
authors.forename: Sanjeev K
authors.initials: SK
authors.lastname: Munshi
authors.affiliation: From Screening and Protein Sciences, Merck Research Laboratories, North Wales, Pennsylvania 19454 and.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Lumb
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D054771:G-Protein-Coupled Receptor Kinase 4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006973:Hypertension / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D054771:G-Protein-Coupled Receptor Kinase 4; C517855:GRK4 protein, human
keywords: G-protein-coupled receptor (GPCR); GRK4; dopamine D1 receptor; hypertension; kinetics; phosphorylation; serine/threonine protein kinase; x-ray crystallography