1308 Characterization of ARC interacting protein, a novel protein that interacts with ARC product which is a novel IEG specific to neuron

Abstract

Northern blot and ribonuclease protection assay were used to identify α2-adrenoceptor subtypes in human colonic adenocarcinoma (HT29), neuroblastoma × glioma rat-mouse hybrid NG108-15 (NG108) and opossum kidney (OK) cell lines. Radioligand binding studies showed that the α2-adrenoceptor expressed in HT29, NG108 and OK cells represent the pharmacological α2A, α2B and α2C subtypes respectively. In our Northern blot analysis, hybridization of poly(A)+ RNA from H129, NG108 and OK cells with human kidney α2-adrenoceptor cDNA probe (α2-C4) identified a single band of 4.4, 4.2 and 4.4 kb respectively in each cell line. Hybridization with a human platelet α2-adrenoceptor genomic probe (α2-C10) resulted in two bands for HT29 cells with the size of 4.4 kb and 3.9 kb. No bands were seen for HT29, NG108 and OK cells when hybridized with a third α2-adrenoceptor human genomic DNA probe which is localized in chromosome 2 (α2-C2). For the HT29 cells, the 3.9 kb band was seen only when using the α2-C10 probe. Thus, this band probably represents α2-C10 mRNA. To further characterize the α2-adrenoceptor mRNA expressed in HT29, NG108 and OK cells, the sensitive ribonuclease protection assay was performed. A single band about 900 bp was protected when the poly(A)+ RNA from NG108 and OK cells was hybridized with an α2-C4 RNA probe and digested with RNAases. Hybridization of mRNA from HT29 cells with α2-C10 RNA probe and digestion with RNAases protected a 500 bp fragment. No bands were seen when the poly(A)+ RNA of these three cell lines hybridized with the α2-C2 RNA probe and digested with RNAases. In conclusion, Northern blot and ribonuclease protection assay in this study provided evidence that the α2-C4 is related to the α2B pharmacological α2-adrenoceptor subtype and is actively transcribed in OK and NG108 cells. The α2-C10 is related to the α2A pharmacological subtype and is actively transcribed in HT29 cells.