G Protein-Coupled Receptor Kinase 5 Contains a DNA-Binding Nuclear Localization Sequence

Abstract

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 ⌬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 ⌬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.G protein-coupled receptor kinases (GRKs) comprise a family of seven serine/threonine protein kinases that phosphorylate agonist-bound, activated, G protein-coupled receptors (GPCRs). GRK-mediated GPCR phosphorylation initiates ␤-arrestin binding, receptor uncoupling from the G protein, and targeting of the ␤-arrestin-receptor complex to a clathrin-coated pit for internalization. The receptor may then be degraded or returned to the cell surface for a further round of signaling (reviewed in reference 4).Seven mammalian GRKs have been identified, which are divided into three subfamilies on the basis of sequence homology and the regulatory mechanisms controlling their activity (reviewed in reference 24): the GRK1-like subfamily, GRK1 (or rhodopsin kinase) and GRK7; the GRK2-like subfamily, GRK2 (␤-adrenergic kinase) and GRK3 (␤-adrenergic kinase 2); and the GRK4-like subfamily, GRK4, GRK5, and GRK6. Four splice variants of GRK4 (␣, ␤, ␥, and ␦) and three splice variants of GRK6 (A, B, and C) have been identified (24).GRKs are regulated by several mechanisms, including modulation of their subcellular localization, kinase activity, and expression (reviewed in reference 21). In many instances, regulation of GRK activity is subfamily specific. PKC phosphorylation results in activation of GRK2 but inhibition of GRK5 activity (21). Additionally, the three GRK subfamilies display differential affinities for calcium binding proteins. GRK1 binds Ca 2ϩ /recoverin, and GRK4␣, GRK5, and GRK6A, -B and -C bind Ca 2ϩ /calmodulin (Ca 2ϩ /CaM) with high affinity (reviewed in reference 32). In contrast, GRK2 exhibits an approximately 40-fold lower affinity for C...

Involvement of Nuclear Factor κB (NF-κB) Signaling Pathway in Regulation of Cardiac G Protein-coupled Receptor Kinase 5 (GRK5) Expression

“…Moreover, the nuclear accumulation of GRK5 in myocytes was in direct result to Gq activation (5). Interestingly, PKC activation by PMA has also been shown to induce GRK5 nuclear localization (4,6).…”

“…G protein-coupled receptors (GPCRs) 4 , such as ␤-adrenergic receptors (␤ARs), play crucial signaling and functional roles in the heart. GPCRs undergo nodal regulation following agonist activation triggered by phosphorylation via family of kinases such as GPCR kinases (GRKs) (1,2).…”

Regulation of Nuclear Factor κB (NF-κB) in the Nucleus of Cardiomyocytes by G Protein-coupled Receptor Kinase 5 (GRK5)

“…GRK5 is unique compared with GRK2 as it has a functional nuclear localization signal (NLS) (9), and indeed, it has been found in the nucleus of myocytes (6,8,10,11). In fact, nuclear localization has been shown to be the determinant of cardiomyopathy after pressure overload where it has non-GPCR activity as a class II histone deacetylase kinase driving cardiac hypertrophic gene transcription (8,12).…”

The G-protein-coupled receptor kinase 5 inhibits NFκB transcriptional activity by inducing nuclear accumulation of IκBα

“…The catalytic domain of GRKs is relatively well-conserved among the members of different subfamilies (Ϸ45% sequence identity), whereas N-terminal RH domains display weak homology (Ϸ27%) and C termini have little or no sequence homology. GRKs have different tissue distribution, subcellular localization, and kinase activity regulation (7,8). GRKs mostly localize at the plasma membrane (2), but recently Johnson et al (7) demonstrated that GRK4-6 (but not other GRKs) can shuttle between cytosol and nucleus through functional nuclear localization sequence (NLS) and nuclear exporting sequence (NES), thus suggesting a nuclear effect for the GRK4-6 subfamily.…”