Studying The Interaction Between ABIN1and MOR,and The Effects Of ABIN1 On The Function Of MOR

Opiates are commonly used for the center analgesia in clinic. Opiates have not only the pharmacological effect such as analgesic, cough-relieving, antidiarrhea, anesthetic, but also have a strong addiction and tolerance. It is easy for opiates to produce dependence with long-term use. For a long-term, scienctists try to investigate the mechanism of opioid addiction from all kinds of aspects, but do not yet clarify its crucial point or find specific solutions to cure the dependence. With the development of molecular biology, the focus of drug research and development gradually change from the screening of discovery of the drug target . Seeking the protein which can interact with the opioid receptor and observing its effect on the development of dependence course may be a meaningful exploration.The aim of previous work in our laboratory was to detect protein-protein interaction. We construted the brain cDNA library originating from morphine dependent rat. Applying the C-terminal ofμ-opioid receptor(MOR) as bait, we screened the brain cDNA library with the bacterial two-hybrid system and acquired 19 positive clones. After analysis, one of positive clone, ABIN1(A20 binging inhibitor of NF-κB activation)is selected for further study. ABIN1 can link A20 to ubiquitinated NEMO which facilitate de-ubiquitination of NEMO mediated by A20 and can inhibit the function of NF-kB. Moreover,ABIN1 is a sensilla of negative regulatory protein in cell death signaling pathway that can inhibit cell aptosis and maintain the embryo growth by combination with polyubiquitin chain. However, it has not been reported about the effect of ABIN1 on the nervous system and on the opioid dependence until now.Preliminary, We must validate the interaction between ABIN1 and MOR because of some possible false positives in two-hybrid system. It is necessary to choose other methods to authenticate the interaction before the next examintaion. On the basis of the exsit of the interaction, we will contiune to study the function of ABIN1.ObjectiveTo validate the interaction between ABIN1 and MOR and to study the effects of ABIN1 on MOR preliminary.Methods1 Constructe the recombinant plasmid for prokaryotic expression, express fussion protein induced by IPTG and validate the interaction between ABIN1 and MOR in vitor by pull-down assay.2 Constructe the recombinant plasmid for eukaryotic expression and transfect transiently CHO cells by liposome-mediated method. Observe the protein distribution of ABIN1 ( aa367-636 ) / ABIN1 ( aa380-636 ) and MOR in cells using immunofluorescence test and validate the interaction between ABIN1 and MOR in vivo by immunoprecipitation assay.3 Utilize KLH as coupling peptide to immunize new zealand rabbit and obtaine antiserum against ABIN1 for subsequent research.4 Constructe stable cell lines cotransfected by ABIN1 and MOR based on the stable cell lines of CHO-μ-68. Acquire stably transfected cells with the high-expression volume by screening polyclonal antiserum and continue the following functinal study.5 Apply the radioligand receptor binding assay and [35S]GTPγS binding assay to investigate the effect of ABIN1 on affinity of the MOR and its ligand and on receptor agonist activity of MOR. Use LANCE? cAMP 384 Kit to detect the effect of ABIN1 on the concentration of cAMP induced by DAMGO.Results1 Pull-down experiments show that fusion protein GST-C can precipitate fusion protein his-hABIN1 specificly and vice versa. The result illustrates the affirmatory existence of the interaction between ABIN1and C-terminal of MOR.2 The results of confocal microscopy reveal that protein ABIN1(aa367-636) / ABIN1(aa380-636) showed a diffuse distribution in the cytoplasm and near the cellmembrance that is similar to MOR arrounding the cell membrane. The phenomenon of co-localization betweenABIN1and MOR provides the possibility of their interactions. Co-immunoprecipitatinexperimental result also further supports the existence of the interaction betweenABIN1 and MOR. These outcomes offer the foundation for study on the function of ABIN1.3 We apply KLH as coupling peptide to immunize new zealand rabbit and obtaine anti-serum. The specificity of antiserum against ABIN1 was characterized by ELISA, Western blot and immunofluorescence. The result displayed that antiserum from rabbitⅡagainst protein ABIN1 is specific.4 We use antiserum from rabbitⅡagainst protein ABIN1 on stably transfected ABINI-MOR cell in western blot assay to identify positive clones and screen the clones with high expression volume. Finally we gain two monoclonals CHO-μ68-ABIN1-19 and CHO-μ68-ABIN1-18 for subsequent experiment.5 We observe the effect of ABIN1 on affinity of the MOR and its ligand with [3H]Diprenorphin binding assay. The results show that the value of Kd and Bmax was 0.19±0.29 nmol/L and 1.71±0.75 pmol/mg protein in CHO-μ-68 cell line as a control, respectively. The value of Kd and Bmax in CHO-μ68-ABIN1-18 cell line has no significant difference compared with that of in CHO-μ-68 cell line. The value of Kd increased 44% and the value of Bmax decreased 64% in CHO-μ68-ABIN1-19 cell line compared to CHO-μ-68 cell line. These results illustrate the decreasing tendency of the maximum binding capacity of receptor and the affinity between MOR and its ligand (P>0.05). We consider that the lower interference of ABINI on affinity of the MOR and its ligand in CHO-μ68-ABIN1-19 cell line caused no significant difference because of the larger standard deviation.6 In [35S]GTPγS binding assay, the EC50 of DAMGO was 3.45 nmol/L in CHO-μ-68 cell line as a control. The value of EC50 in CHO-μ68-ABIN1-18 cell line has no significant difference compared with CHO-μ-68 cell line. The value of EC50 in CHO-μ68-ABIN1-19 cell line showed decreasing tendency when compared to CHO-μ-68 cell line(P>0.05). These results accordant with the results of [3H]Diprenorphin binding assay.The interference of ABINI on affinity may also affect the activty of MOR.7 Forskolin (10μmol/L) can activate protein Gs and significantly increase the concertration of cAMP in CHO-μ68-ABIN1-19 cell lines ,CHO-μ68-ABIN1-18 and CHO-μ-68 cell lines, while DAMGO can significantly inhibit this increasing induced by forskolin owing to its activation of protein Gi. In CHO-μ68-ABIN1-19 cell lines and CHO-μ68-ABIN1-18, the concertration of cAMP shows a trend of the decreasing trendency compared to CHO-μ-68 cell lines, but there is no significant difference (P> 0.05).These results hint that ABIN1 may affect post-receptor signaling pathway of MOR, but the effect is not obvious. ConlusionWe validate the existence of interaction between ABIN1and C-terminal of MOR by pull-down assay, immunofluorescence co-localization detection andimmunoprecipitation examination. With the function determination of ABIN1 on receptor binding, receptor agonist activity and post-receptor signaling molecules , We can draw our conclusion that ABINI may affect the function of MOR to some extent, butdefinitive conclusions need yet to be further studied.