Screening And Verification Of The Intracellular Proteins Interacting With The C-terminal Of μOpioid Receptor

ObjectiveOur study was designed to identify and characterize novel intracellular signal molecules which specially mediated the phosphorylation and desensitization of MOR when MOR was activated continuously. With the help of the novel intracellular signal molecules, the biochemical and molecular mechanisms of opioids tolerance and dependence were further explored, which might lead a new direction for the study of opioids dependence.Content and MethodsBacterioMatch(?) two-hybrid system is an efficient tool for detecting protein-protein interactions in vivo. We constructed the brain cDNA library through reverse transcription and LD-PCR based on mRNA of morphine dependent rat. Detection of protein-protein interactions is based on transcriptional activation of the HIS3 reporter gene, which allows growth in the presence of 3-AT, a competitive inhibitor of HIS3 enzyme on his-dropout selective screening medium. A second reporter gene, aadA, encording streptomycin, provides a mechanism to validate the bait and target interaction. The C-terminus ofμ-opioid receptor as a bait was fused toλ. repressor protein(λcI) on plasmid pBT . We screened about 3×10~6 cotransformants and sequenced the positive interaction clones. The pull-down test confirmed the interaction of novel proteins with MOR-C in vitro. The protein-protein interactions were also tested by immunocytochemistry and coimmunoprecipitation.Results1 We constructed the brain cDNA library of morphine dependent and normal rat .The capacity of the library was over 10~6 cfu with about 1kb cloned segment. The recombination rate of the library was above 90% and it was suitable for the library screening.2 Using the MOR-C as bait, we screened the brain cDNA library with the Bacterial two-hybrid system. Total 154 positive clones which were harvested from the selective screening medium and 124 positive clones from the dual selective screening medium were obtained, respectively. After the cotransformation of the cDNA plasmid with pBT or pBT-C, 19 positive clones were acquired.3 The sequences of the 19 positive clones were aligned with NCBI BLAST nr database to glean information about the molecular targets. The proteins interacted with MOR-C are as follows: p75NTR-associated cell death executor (NADE), TNFAIP3 interacting protein (ABIN1) , cystatin C, insulin-like growth factor, heat shock protein , zinc finger protein 216, mitochondrion, chromosome gene et al. Additionally, there were 5 unrecognized proteins.4 The interaction of NADE and ABIN1 with MOR-C was confirmed by in vitro protein-protein binding. To verify that direct interaction may exist between NADE / ABIN1 and MOR, column overlay experiments were performed. GST fusion proteins of MOR-C (GST-C), and the hexahistidine fusion protein of the C-terminal portion of NADE/ABIN1 were generated, as described in the experimental procedures.5 In order to determine whether co-localization exists in CHO cells between NADE, ABIN1 and MOR, double immunofluorescent staining was performed in CHO cells containing both HA -MOR and GFP- NADE/ABIN1 by using anti-HA (red) and fluorescently labeled secondary antibodies, as described in the experimental procedures. With the help of Confocal microscopy, significant co-localization (yellow) was observed on the cell membrane. Binding between MOR and NADE/ABIN1 was further supported by immunoprecipitation studies. We found that precipitation of HA-MOR with HA antibodies resulted in the coprecipitation of myc-NADE/ABIN1.ConclusionsThe screening with the bacterial two-hybrid system, using the C-tail of MOR as bait and morphine dependent cDNA library as target, indicated that NADE, a p75NTR-associated cell death executor and ABIN1, a TNFAIP3 interacting protein interact with the C-tail of MOR. The results from the overlay experiments, immunofluorescent studies and co-immunoprecipitation studies confirmed the binding between the MOR-C and NADE/ABIN1 both in vitro and in vivo. This study constructed the brain cDNA library of the morphine dependent rat for the first time and there are no reports about the interaction between MOR-C and NADE/ABIN1 until now. It need further study for the effects of NADE/ABIN1 on the regulation of MOR function.