Mechanism Of Nuclear Translocation Of G Protein-coupled Receptor Kinase 6

Objectives:G protein-coupled receptor kinases（GRKs）phosphorylate the ligand activated G protein-coupled receptors（GPCRs）and negatively regulate GPCRs signaling pathways.Recently,it has been reported that several GRKs including GRK6 directly regulate nuclear signal molecules.Obviously,nuclear translocation is necessary for the nuclear regulation of GRKs.However,the mechanism of nuclear translocation of GRKs,including GRK6,is largely unclear.One of the aims of this study is to identify the functional nuclear localization sequences（NLSs）of GRK6 and the crucial residues of NLS,another is to identify the karyopherins(（KPNs or Importins,IPOs）that involved in the nuclear transportation of GRK6.Methods:To screen the NLSs of GRK6 and the crucial residues,NLSs of GRK6 were predicted by PredictNLS software and the putative NLSs were deleted or substituted alkaline amino acids with alanine.The mutants were expressed in HEK293 cells.Then,cells expressed these mutants were subjected to immunofluorescence microscopy and cell fractionation assay to determine the subcellular distribution of GRK6 mutants.Next,to detect the crucial residues whether have a negative affection on its kinase activity,cells stably expressing the GKR6 WT or mutants which crucial residues were substituted with alanine were stimulated by agonist（carbacol）of M3 receptor and the mobilization of intracellular calcium induced by the activated M3 receptor was detected by calcium assay kit.Ten karyopherins candidates were screened by RNA interference（RNAi）and microscopy.Then,we employed bimolecular fluorescence complementation（BiFC）assay and Co-Immunoprecipitation（Co-IP）assay to detect the interaction between GRK6 and karyopherin.Results:Site mutations in K³⁸⁹,K³⁹⁰,K³9191 of GRK6 show a significant inhibition on its nuclear distribution but do not destroy the inhibition ability on the mobilization of intracellular calcium induced by the activated M3receptor;Only importin-11 knockdown showed an inhibition of nuclear localization of GRK6 while the other nine karyopherins were not when compared with the control group.Conclusion:K³⁸⁹,K³⁹⁰,K³99 are crucial for the nuclear localization of GRK6,which is mediated by importin-11,and the mutations in this residues do not destroy its kinase activity.