The Effects Of ABIN1on The Function Of μ Opioid Receptors

Drug abuse including opium was a worldwide problem. Morphine, a major component of opium, has a wide rangeof effects in the body, but the most important one is the relief of pain. Opioids drugs caused serious side effects in central nervous system, especially tolerance and addiction with long-term use, which limited the clinical application and become a serious international problem. The studies on opioid tolerance and addiction showed that, with long-term stimulation from opioid drugs, the expression of opioid receptor was down regulated, the active state and the ligand binding ability of opioid receptor were changed, and many down-stream signals were influenced. These are known as a possible mechanism of opiod drugs tolerance and addiction. The process of tolerance and addiction is very complicated, and its molecular mechanisms are still unknown. The main work in our laboratory is to look for the proteins which may interact with MOR and its interacting mechanisms, which may show a new therapeutic target for the treatment ofopioid dependence. Using A bacterial two-hybrid system, the C-terminal of MOR was found to be associated with19proteins includingABIN1(A20-binding inhibitor of NF-kB). ABIN1physically links A20to ubiquitinated NEMO, thus facilitating A20-mediated de-ubiquitination of NEMO and NF-kB inhibition.. The activation of opiod receptor can inhibit the function of NF-kB. The interaction between ABIN1and MOR was demonstrated by pull-down of induced protein expression, immunofluorescence and Co-IP in ABIN1and MOR cotransfected cells in our laboratory previously. Little was known about the effects of ABIN1on the fuction of MOR.The Objectives are:To study the effects of ABIN1on μ-opioid receptor signal transduction and to demonstrate the changes of ABIN1in the central system of the morphine dependent rats and mice. In the frist part:Expression and the colocalization of ABIN1and MOR proteins in the stable cell lines cotransfected by pcDNA3.1-ABIN1and pCMV6-myc-flag-MOR.(1) The results of western-blotting revealed that MOR was expressed by μ-CHO-68and μ-ABIN1-CHO-19cell line, while ABIN1was expressed by μ-ABIN1-CHO-19cell line.(2) ABIN1and MOR were colocalized in the cell membrane. Of CHO by confocal microscopy; Effect of ABIN1on the signal transduction of MOR when it was agitated by DAMGO.(1) In [35S] GTPyS binding assay, in μ-CHO-68cell lines, the EC50of DAMGO was31.10±12.50(nmol/L) and the Emax6.34±0.9, compared with EC50121.71±44.28(nmol/L) in μ-ABIN1-CHO-19cell lines which was significantly different from μ-CHO-68cell lines (P<0.01). The Emax was4.56±1.25in μ-ABIN1-CHO-19cell lines which showed decreasing tendency when compared to μ-CHO-68cell lines though has no significance.(2) The phosphorylation of MOR induced by DAMGO were decreased significantly in μ-ABIN1-CHO-19compared with μ-CHO-68cell lines.(3) ABIN1inhibited the endocytosis of MOR significantly in μ-ABIN1-CHO-19compared with μ-CHO-68cell lines when the MOR were agitated by DAMGO by confocal microscopy.(4) ABIN1decreased the inhibition of DAMGO on the forskorlin-stimulated cAMP over-shoot. After chronic morphine exposure, ABIN1inhibited naloxone-precipited Ca2+overshoot.(5) The phosphorylation of ERK was down-regulated by application DAMGO in μ-ABIN1-CHO-19cells compared with μ-CHO-68cells.In the second part:Preparation of antiserum against Rat-ABIN1. Distribution of ABIN1on the rat central nervous system..(1) Using KLH as coupling peptide, immunized new-zealand rabbit and obtained antiserum. The specificity of antiserum against ABIN1was characterized by ELISA, Western blotting and immunofluorescence. The result displayed that antiserum from341against protein ABIN1of rat is specific. Purification of the antiserum made its valency low but more specific.(2) The results of Western-blotting and qPCR revealed that ABIN1was distributed in encephalic region of normal rat widely and highly; The variation of ABIN1on the central nervous system of the morphine dependent rats and mice.(1) With increasing doses, rats were administrated with morphine continuously for30days to induce dependence. Expression of ABIN1began to increase14days after morphine administration and the most significant up-regulation of ABIN1was31-37days during the natural withdrawl. ABIN1was restored to control level during prolonged withdrawl time.(2) ABINI increased significantly in morphine dependent and naloxone precipitated procortex, hippocampus and striatum corpora of mice.ConlusionABIN1inhibited the activity of MOR and its signal transduction significantly. ABIN1increased obviously in different brain region of morphine dependent rats and mice.