title: The regulator of G protein signaling (RGS) domain of G protein-coupled receptor kinase 5 (GRK5) regulates plasma membrane localization and function.
abstract: The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated GPCRs at the plasma membrane (PM). Here GRK5/GRK4 chimeras and point mutations in GRK5 identify a short sequence within the regulator of G protein signaling (RGS) domain in GRK5 that is critical for GRK5 PM localization. This region of the RGS domain of GRK5 coincides with a region of GRK6 and GRK1 shown to form a hydrophobic dimeric interface (HDI) in crystal structures. Coimmunoprecipitation (coIP) and acceptor photobleaching fluorescence resonance energy transfer assays show that expressed GRK5 self-associates in cells, whereas GRK5-M165E/F166E (GRK5-EE), containing hydrophilic mutations in the HDI region of the RGS domain, displays greatly decreased coIP interactions. Both forcing dimerization of GRK5-EE, via fusion to leucine zipper motifs, and appending an extra C-terminal membrane-binding region to GRK5-EE (GRK5-EE-CT) recover PM localization. In addition, GRK5-EE displays a decreased ability to inhibit PAR1-induced calcium release compared with GRK5 wild type (wt). In contrast, PM-localized GRK5-EE-CaaX (appending a C-terminal prenylation and polybasic motif from K-ras) or GRK5-EE-CT shows comparable ability to GRK5 wt to inhibit PAR1-induced calcium release. The results suggest a novel model in which GRK5 dimerization is important for its plasma membrane localization and function.
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107.
authors.forename: Hua
authors.initials: H
authors.lastname: Xu
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107Center for Science Research, Guilin Medical University, Guilin, Guangxi 541004, China jiangxs@glmc.edu.cn philip.wedegaertner@jefferson.edu.
authors.forename: Xiaoshan
authors.initials: X
authors.lastname: Jiang
authors.affiliation: Center for Science Research, Guilin Medical University, Guilin, Guangxi 541004, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Shen
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Fischer
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107 jiangxs@glmc.edu.cn philip.wedegaertner@jefferson.edu.
authors.forename: Philip B
authors.initials: PB
authors.lastname: Wedegaertner
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology*; D054774:G-Protein-Coupled Receptor Kinase 5 / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D021381:Protein Transport
chemical_list: D054774:G-Protein-Coupled Receptor Kinase 5; C517898:GRK5 protein, human