The Role Of Grk In The Cell Cycle And Neuronal Development

G protein-coupled receptor kinases act as negtive regulators of GPCR signals.It has been reported recently,GRK can bind with nonreceptor substrates,except for phospholation and desensitization of GPCR,showing multiple functions.GRK2 and GRK5 are members of GRKs family.Our Previous research has shown GRK2 can bind EGFR,but the function is unclear,Previous research has shown that cells were arrested in mitosis after GRK5 was knocked down,the underling mechanism is not clear;GRK5 expressed highly in some certain stage of neuron development,but the function of GRK5 in the process is not clear. Our gene screen results showed that genes of cell cycle and neuron development upregulated after GRK5 was knocked down.Here research focuses on the role of GRK2 in signal crosstalk and physiological functions of GRK5 on cell mitosis and neuron development.The underling mechanism also is included.In research of GRK2 function in signal crosstalk,rusults show that:1)By the stimulation of EGF,GRK2 translocates to cell membrame.2)EGF pretreatment increases DOR endocytosis mediated by GRK2.In research about GRK5 function in cell mitosis,rusults show that:1)p-H3 positive and Tunel positive cells increase after GRK5 RNAi,which suggests that knock down of GRK5 arrests cells in metaphase and induces cell apoptosis.2)Using DAPI labeling chromosome, we find that deletion of GRK5 induces chromosome misalignment but has no effect on cytokinesis.3)Living cells observation shows that,after deletion of GRK5,cells fail to align chromosomes to the equatorial metaphase plate and can't go throughout the cell cycle, leading to apoptosis.Our results first show that knock down of GRK5 induces chromosome misalignment and cells are arrested in metaphase,leading to apoptosis.In research of GRK5 function on neuron development,results show that:1)overexpression of GRK5 in neuron cultures promots neurite outgrowth.Furthermore overexpression of GRK5 kinase dead mutant,K215R,induces neurite outgrowth too.It is suggested that, GRK5 can promote neurite outgrowth,which is kinase independent.2)GRK5 colocalizes with actin in growth cones and bingding F-actin.GRK5 promotes bundling of F-actin.The actin binding domain mutant GRK5_C5Mcan't induce neurite outgrowth.It is showed that GRK5 regulates neurite outgrowth by bindling F-actin.3)In hipocampal neuron cultures, dominactive Racl,dominactive cdc42 and constitutive active RhoA can inhibit the neurite outgrowth induced by GRK5.It is suggested that Rho GTPases are involved in this progress. 4) In utero electrocoporation experiment,deletion of GRK5 in cortical neurons induces less and shorter branches compared with control.Our results suggest that knockdown of GRK5 in cortical neurons prevents development of dendritic tree,but has no effect on the migration and polarization of neuron progenitors.5) In knock out mice,spines of hipocampal neurons show abnormal morphology,slim and longer with smaller ration of head/neck.In a word,our data first show that:1)GRK2 mediates the regulation of EGF signal on the DOR endocytosis;2) GRK5 regulates cell cycle by maintaining chromosome alignment:3) GRK5 rugulates neurite outgrowth by bundling actin.GRK5 is required for dendrite morphogenesis and spine morphology maintenance in vivo.Current study uncovers the novel functions of GRK2 in signal crosstalk,the functions of GRK5 in cell cycle and neuron development.