G protein-coupled receptor kinase 5 contains a DNA-binding nuclear localization sequence.
G protein-coupled receptor kinases (GRKs) mediate desensitization of agonist-occupied G protein-coupled receptors (GPCRs). Here we report that GRK5 contains a DNA-binding nuclear localization sequence (NLS) and that its nuclear localization is regulated by GPCR activation, results that suggest potential nuclear functions for GRK5. As assessed by fluorescence confocal microscopy, transfected and endogenous GRK5 is present in the nuclei of HEp2 cells. Mutation of basic residues in the catalytic domain of GRK5 (between amino acids 388 and 395) results in the nuclear exclusion of the mutant enzyme (GRK5(Delta)(NLS)), demonstrating that GRK5 contains a functional NLS. The nuclear localization of GRK5 is subject to dynamic regulation. Calcium ionophore treatment or activation of Gq-coupled muscarinic-M3 receptors promotes the nuclear export of the kinase in a Ca(2+)/calmodulin (Ca(2+)/CaM)-dependent fashion. Ca(2+)/CaM binding to the N-terminal CaM binding site of GRK5 mediates this effect. Furthermore, GRK5, but not GRK5(Delta)(NLS) or GRK2, binds specifically and directly to DNA in vitro. Consistent with their presence in the nuclei of transfected cells, all the GRK4, but not GRK2, subfamily members contain putative NLSs. These results suggest that the GRK4 subfamily of GRKs may play a signaling role in the nucleus and that GRK4 and GRK2 subfamily members perform divergent cellular functions.
Johnson
Laura R
LR
Laboratory for Molecular and Cellular Biology and Department of Pharmacology, University College London, Gower St., London WC1E 6BT, United Kingdom.
Scott
Mark G H
MG
Pitcher
Julie A
JA
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002118:Calcium / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D054774:G-Protein-Coupled Receptor Kinase 5; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D012313:RNA / Q000737:chemistry; D043587:Receptor, Muscarinic M3 / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014162:Transfection
D002147:Calmodulin; D018076:DNA, Complementary; D007476:Ionophores; D043587:Receptor, Muscarinic M3; D012313:RNA; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D054774:G-Protein-Coupled Receptor Kinase 5; C517898:GRK5 protein, human; D002118:Calcium