Identification Of Rat GR MRNA Spliced Variant And Study On GR MRNA Expression

Glucocorticoid receptor (GR), together with mineralocoroticoid receptors (MR), androgen receptors (AR), estrogen receptors (ER),and progesterone receptors (PR),as members of the intracellular receptor superfamily of transcriptional regulatory proteins, belongs to the steroid receptor subfamily. It was widely accepted that GR as a typical hormonal dependent transcription regulation factor regulated gene transcription by binding as a homodimer to specific GC response elements (GREs) in GR target genes, and ultimately caused a variety of biological effects. This was genomic effect of GC, and it belonged to its classic role mode. Recent research showed that GC not only directly but indirectly regulated gene transcription and exerted physiological and pharmacological functions. However, recent evidence indicated that GC might also act via non-genomic actions to exert multiple rapid effects such as hormone secretion, neuronal excitability, behavior, cell morphology, carbohydrate metabolism and other processes within seconds or minutes. Because these effects happened in a short period of time, this was called the rapid effect. Even though the mechanism of the GC's rapid effect was not clear, the non-genomic effect might depend on specific receptor or liposolubility of the GC itself. More and more reports showed that GC might exert different effect by action with different GR spliced variant.Since the GR mRNA sequence of rat was cloned in 1985, some reports assumed the presence of other GR spliced variant. And several GR proteins extracted from the different tissue of rats and mice have been successfully characterized. Even though it might present different molecular weigh of GR from the protein level, no novel spliced variant of GR mRNA was reported. In the study of the interaction between regulator of G protein signaling 4(RGS4) and GR domains by using two yeast hybrid system, we found a new GR mRNA sequence. Compared with the reported GR mRNA's sequence, the new GR spliced variant sequence was 42 bp shorter. The absence region located in transcription regulation region and encoded repetitious Gln-glutamine. The newly GR short mRNA might be a new spliced variant of GR mRNA.Two couples of primer were designed based on non-homology region of the GR long and short sequence. After amplifying the GR-long and short fragment with the outer primer from the rat genomic DNA and pGAD-T7-sAB, the probes amplified with the biotin-labeled inner primer, and the PCR production of the first step as mould. The size of the production analyzed by agarose gel electrophoresis (AGE) and polyacrylamide gel electrophoresis (PAGE). The quantity and distribution of GR long and short mRNA in normal tissues of rat was detected by Dot Blot and Northern Blot hybridization assay. The results of Dot blot showed that the GR short mRNA distribution as follow, lung>brain>kidney>liver; and the GR long mRNA distributed most abundant in brain and least in liver, almost the same quantity in lung and kidney. The results of Northern Blot assay showed that the new and old GR mRNA was detected in a different tissue; a band of 6.3 kb corresponding to GR mRNA was identified in all tissues. The distribution of GR long mRNA was as follows: heart