Preliminary Research On The Biological Characteristics And Function Of IRAS, A Candidate For â… ₁-imidazoline Receptor

Our previous work proved I₁-imidazoline receptor to be a new important target which modulated opioid dependence and proposed that agmatine and imidazoline receptors consisted of a novel system of modulating opioid functions. The present study is to focus on the biological features and function of IRAS(imidazoline receptor antisera selected protein), a candidate protein of I₁-imidazoline receptor and to elucidate the molecular identity. The results presented here facilitate further research on agmatine molecule mechanism. Firstly, based on the bioinformatics analysis, the 43.1a-IRAS(10-120aa)protein was expressed in the prokaryotical system and purified. Hybridoma cell lines that secreted anti-IRAS mAb were established. Reaction of mAb to IRAS protein was characterized by Western blot, indirect immunofluorescent staining, immunoprecipitation, immunohistochemical staining and FCM. It was found that the established antibodies reacted specifically with IRAS.Using a specific antibody of IRAS, IRAS protein were quantitatively and qualitatively detected and the cellular localization of IRAS was further confirmed. As a powerful reagent for the study of IRAS, the results presented here summarized for the first time the expression and distribution of the IRAS in different tissues by immunohistochemical, Western blot and studied the subcellular localization of IRAS by immunofluorescence. We preliminarily studied the signal transduction pathway of IRAS which participated in cell proliferation and survival and also observed IRAS expression associated with cell cycle progression. We demonstrated that (1) Interestingly, the expression of wild IRAS was only found in the liver. Surprisingly, the most prominent expression of IRAS was a specific band between 47-60KDa in other tissues. IRAS was also localized mainly in the cytoplasm, suggesting the expression of IRAS was tissue-specific and variable (2) The function of IRAS was associated with its cellular localization, and wild IRAS exhibited targeting to punctate structures in mammalian cells. The distribution of IRAS was dependent on the presence of a PX domain at its NH₂ terminus. With truncation of the PX domain, IRAS exhibited cytosolic distribution (3) IRAS's intrinsic activity was to promote cell growth and survical, we revealed that a signaling function for IRAS lay in activation of extracellular signal-regulated kinase(ERK), P38MAPK, and PI₃-kinase (4) We cloned new alternative spliced variants of IRAS and believed the variety of IRAS tissue expression related to expression of alternative spliced variants. In conclusion, based on the IRAS gene and specific monoclonal antibody of IRAS, we provided new insight into IRAS's candidacy as an I₁-R.