Isolation Characterization And Expression Of CDNA Clones Encoding A G Protein-coupled Receptor Kinase Of The Ascidian Ciona Intestinalis

ObjectiveG protein-coupled receptors (GPCRs) mediate cellular responses to a diverse range of extracellular stimuli. G protein-coupled receptor kinases (GRKs) are responsible for both Agonist-induced desensetization and resensetization of GPCRs, Although many kinds of GRKs have been isolated from both vertebrates and invertebrates, little is known about the role and regulation of GRKs in the process of G protein signalling. In an effort to carry out a molecular genetic dissection of the role and regulation of G protein-coupled receptor kinase (GRK), we processed the research of the GRK of the ascidian dona intestinalis, one of the most primitive extant chordate relatives of the vertebrates, in order to get some essential information of the same but more complicated system of the vertebrates.MethodsFisrtjotal RNAwas prepared using Isogen kit. By RT-PCR, 3'RACE and so on, we isolated cDNA clones encoding GRK from the ascidian dona intestinalis, Then GENETYX-MAC/ATSQ software and CLUSTAL W program were used for the sequence analysis. At last, we performed RT-PCR using total RNA of different development stages as the DMA template to determine at which stage of the development the gene are expressed.Results1 We isolated cDNA clones encoding GRK from the ascidian Ciona intestinalis, and designated the corresponding gene as Ci-GRK1.2 Analysis of the complete nucleotide sequences of the cDNA clones revealed that the open reading frame for Ci-GRKI encodes 542 amino acids and contains all of the amino acids that are highly conserved in protein kinase family and in GRKs family.3 Molecular phylogenetic analysis shows Ci-GRK1 is as a member of the GRK4/5/6 subfamily.4 Ci-GRKI shows the highest amino acid sequence identity with human GRK5(66%).5 By RT-PCR analysis, we detected Ci-GRKI transcripts at all stages of embryonic development.ConclusionThese results suggest that Ci-GRK1 is the principal member of the family that is expressed during development. The modulation of GPCR activity by Ci-GRK1 might be important for developmental signal transduction. The high homology between Ci-GRKI and human GRK5 reveals that the research of the expression> differentiation and signal transduction of the ascidian Ciona intestinalis will provide some insight into the complex signal transduction of human.