G protein-coupled receptor kinase 5 contains a DNA-binding nuclear localization sequence
G protein-coupled receptor kinase 5 contains a DNA-binding nuclear localization sequence
复制标题
DOI：
10.1128/mcb.24.23.10169-10179.2004
复制
发表时间：
2004-12-01
影响因子：
5.3
通讯作者：
Pitcher, JA
中科院分区：
文献类型：
--
作者：
Johnson, LR;Scott, MGH;Pitcher, JA
G 蛋白偶联受体激酶 (GRK) 介导激动剂占据的 G 蛋白偶联受体 (GPCR) 脱敏。在这里，我们报道GRK5含有DNA结合核定位序列（NLS），并且其核定位受GPCR激活调节，结果表明GRK5具有潜在的核功能。通过荧光共聚焦显微镜评估，转染的内源 GRK5 存在于 HEp2 细胞的细胞核中。 GRK5 催化结构域中的碱性残基（氨基酸 388 和 395 之间）的突变导致突变酶 (GRK5(DeltaNLS)) 被核排除，证明 GRK5 含有功能性 NLS。 GRK5的核定位受到动态调控。钙离子载体处理或 Gq 偶联毒蕈碱 M3 受体的激活以 Ca2+/钙调蛋白 (Ca2+/CaM) 依赖性方式促进激酶的核输出。 Ca2+/CaM 与 GRK5 N 端 CaM 结合位点的结合介导了这种效应。此外，GRK5（而非 GRK5(DeltaNLS) 或 GRK2）在体外特异性且直接地与 DNA 结合。与它们在转染细胞核中的存在相一致，所有 GRK4（但不是 GRK2）亚家族成员都含有推定的 NLS。这些结果表明 GRK 的 GRK4 亚家族可能在细胞核中发挥信号传导作用，并且 GRK4 和 GRK2 亚家族成员执行不同的细胞功能。
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(DeltaNLS)), 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 Ca2+/calmodulin (Ca2+/CaM) -dependent fashion. Ca2+/CaM binding to the N-terminal CaM binding site of GRK5 mediates this effect. Furthermore, GRK5, but not GRK5(DeltaNLS) 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.