G Protein-coupled Receptor Kinase 4 Expression Pattern And Cloning Of Novel Splicing Variants In Rat Tissues

Objective: G protein-coupled receptor kinases(GRKs) are key regulators that, together with arrestins, determine the rate and extent of homologous desensitization of G protein-coupled receptors(GPCRs) by phosphorylating activated receptors. Subsequent studies revealed that GRK is a family composed of seven isoforms, GRK1 through GRK7. The seven known GRKs have been identified and classified into 3 distinct subfamilies based on sequence and functional similarities. Each GRK shows a differential expression pattern. GRK1,GRK4, and GRK7 are expressed in limited tissues. In contrast, GRK2, GRK3,GRK5, and GRK6 are ubiquitously expressed throughout the body.Comparison of the amino acid sequences of GRKs from various mammalian species indicates that GRK2, GRK5, and GRK6 exhibit a remarkably high degree of sequence conservation, whereas GRK1 and particularly GRK4 have accumulated amino acid changes at extremely rapid rates. The differential tissue distribution of GRK4 also suggests that individual GRK4 variants may serve distinct physiological functions. These peculiar characteristics make GRK4 a unique member within the GRK multi-gene family.Rat GRK4(575 amino acids) splices in a manner analogous to human, and displays 76% identity with the long human GRK4 splice variant. Thus, I selected rat tissues at different ages, study the differential tissue distribution and functional expression of rat GRK4 and its splicing variants in both mRNA and protein level.Methods : 1. GRK4 expression pattern in rat tissues. 1) Extract total RNA from rat tissues at different ages and study rat GRK4 gene expression pattern by quantitative real-time PCR. 2) Extract total protein from rat tissues and study the expression of GRK4 or its splicing variants by western blot. 2. Cloning of GRK4 gene and splicing variants: a. According to the rats GRK4 gene sequences in GenBank design primers; b. Extract total RNA from rat testis and epididymis with Trizol, then amplify them to cDNA by reverse-transcription chain reaction(RT-PCR), amplify GRK4 genes and its splicing variants using specific primers, electrophoresis PCR products; c. Cloning the product cDNA fragments into p MD 18-T vector, then transform them into E.coli DH5α cells to verify by PCR screening and sequencing;Using NCBI-BLAST tool to align DNA sequences and analysis them online.Results: 1. GRK4 expression pattern in rat tissues. 1) Testing GRK4 gene expression pattern by quantitative real-time PCR in mRNA level.(1) GRK4 gene expresses widely in rat tissues.(2) Rat ovary GRK4 gene expression level changes along with the ovary grow and mature. 2) Testing GRK4 expression by western blot in protein level. The result shows that GRK4 expresses highly in rat thymus, testis, epididymis and brain tissue, and the expression may be different at different development stages of rat too. 2. Cloning of rat GRK4 gene and novel splicing variants. The sequencing results were compared with thesequences reported in Gen Bank. The result showed that two novel splicing variants in GRK4 open reading frame, named them GRK4 variant X1 and GRK4 variant X2 respectively.Conclusions: Gene can produce multiple splicing variants by alternative splicing in the process of transcription, translation. This experiment provided a new clue for the research of GRK4 by molecular biological technology such as quantitative RT-PCR, western Blot, and gene cloning. The results indicated that rat GRK4 or splicing variants express highly in testis, thymus and brain. We would clone rat complete GRK4 DNA and other splicing variants, and figure out the structure and function of GRK4 variants in the protein level for further study.