Construction And Expression Of Opioid Receptors Using Eukaryotic Expression System And Investigation In The Interaction Between μ And δ Opioid Receptor

BACKGROUND & OBJECTIVEThe illicit use of drugs such as morphine, heroin has become a social nuisance. It is difficult to cure drug addiction because of severe withdrawal symptoms and high rate of reuse.Drug addiction is a chronic relapsing encephalopathy. The mechanism of drug addiction is complex and involves many brain areas of the central nervous system.Opioid receptors have distinct pharmacological profiles and discrete but overlapping distributions in brain. The endogenous opioid peptide receptor systems mediate important physiological functions related to pain perception, locomotion, motivation, reward, autonomic function, immunomodulation and neuroendocrine function. Pharmacological and molecular cloning studies have identified three opioid receptor types,μ(MOR),δ(DOR)and k(KOR) that mediate these diverse effects.In recent years, with the development of neuroanatomy, neuropharmacology and neural mechanisms of drug dependence, more and more facts show that nucleus accumbens plays a key role in drug dependence. Many animal experiments and anatomical studies have shown that nucleus accumbens is closely related with drug addiction. Bilateral destruction of rat nucleus accumbens can inhibit the establishment of heroin self-administration, eliminate drug-seeking behavior and recrudescence in rats addicted.We speculate that heroin addiction has a significant bearing on protein-protein interaction ofμandδopioid receptors in nucleus accumbens. The inference is based on several facts. Firstly, a lack ofμorδreceptors abolishes the analgesic effect of morphine, as well as place-preference activity, hypolocomotion and physical dependence. Mice withμorδreceptor gene knock out do not develop analgesic tolerance to morphine. Secondly, many experiments have proved that there is a physical interaction betweenμandδreceptors and thus have the potential to create novel signaling units. Thirdly, theδopioid receptor andμopioid receptor are abundantly distributed in the dorsal horn of the spinal cord. Simultaneous activation of each receptor by selective opiate agonists has been shown to result in synergistic analgesic effects. Immunoperoxidase and immunogold-silver labeling methods showed 8 opioid receptors are often in cooperation withμopioid receptors in striatal patches. Fourthly, Schmidt BL showed thatμandδreceptors contributed to capsaicin-induced antinociception in nucleus accumbens; selective activation of individual receptor subtypes was insufficient, but coactivation ofμandδopioid receptors induced antinociception. Fifthly, stereotactic destruction of nucleus accumbens in experimental animals and patients with heroin addiction have achieved good results.Shao-Ping Ji synthesized the interfering peptide Tat-3L4F, which is able to disrupt PTEN coupling with 5-HT2cR. Systemic application of Tat-3L4F suppressed the increased firing rate of VTA dopaminergic neurons induced by Δ9-tetrahydrocannabinol (THC), the psychoactive ingredient of marijuana. Tat-3L4F blocks conditioned place preference of THC or nicotine, and does not produce side effects such as anxiogenic effects, penile erection, hypophagia and motor functional suppression. These results suggested a potential strategy for treating drug addiction with the Tat-3L4F peptide.If we can find the specific peptide sequence for interaction betweenμandδopioid receptors, and thus using the peptide as interfering peptide, which maybe be able to disruptμreceptor coupling withδreceptor, may suppress behavioral responses induced by drugs of abuse such as heroin.METHODS & RESULTSThe total RNA was isolated from nucleus accubems using Trizol according to its instructions. Full length cDNAs of ratμ,δ,κopioid receptor were amplified from the rat brain tissue through reverse transcription and nested PCR, respectively. The full length cDNAs ofμ,δ,κopioid receptor were ligated with pMD20 T vector through A-T ligation, respectively. There was a single site mutation inμ,δ,κopioid receptor, respectively. Site mutation ofμ,δ,κopioid receptor was corrected using Stratagene mutation kit. Full length DNA of ratμopioid receptor tagged with a 10-residue Myc epitope in carboxyl terminal was amplified fromμ-pMD20 T vector by PCR with high fidelity DNA polymerase. Full length DNA of rat 8 opioid receptor tagged with a 8-residue FLAG epitope in carboxyl terminal was amplified from 8-pMD20 T vector by PCR with high fidelity DNA polymerase. Full length DNA of ratκopioid receptor tagged with a 9-residue HA epitope in carboxyl terminal was amplified from K-pMD20 T vector by PCR with high fidelity DNA polymerase.The PCR amplified DNAμ-Myc was purified by PCR clean up kit and attached a 3'dA nucleotide overhang and ligated with pMD20 T vector to formμ-Myc-pMD20 T vector. The PCR amplified DNAδ-FLAG was purified by PCR clean up kit and attached a 3'dA nucleotide overhang and ligated with pMD20 T vector to formδ-FLAG-pMD20 T vector. The PCR amplified DNAκ-HA was purified by PCR clean up kit and attached a 3'dA nucleotide overhang and ligated with pMD20 T vector to formκ-HA-pMD20 T vector.δ-FLAG-pMD20 T vector and pIRES2-EGFP plasmid were digested with EcoRl and BamHI, respectively. DNA fragmentδ-FLAG and pIRES2-EGFP digested by EcoRI and BamHI was isolated using agarose gel DNA recycling kit, respectively. Then they were ligated to formδ-FLAG-pIRES2-EGFP plasmid with T4 DNA ligase. The DNA sequence ofδopioid receptor with FLAG epitope was inserted into pIRES2-EGFP and confirmed by sequencing on both strands and restriction enzyme digestion. At the same time,μ-Myc-pMD20 T vector and pIRES2-EGFP plasmid was digested with EcoRI and NheⅠ, respectively. By the same method of restriction enzyme digestion, isolation using agarose gel DNA recycling kit and ligation using T4 DNA ligase,μ-Myc-pIRES2-EGFP andκ-HA-pIRES2-EGFP were constructed and confirmed by sequencing on both strands and restriction enzyme digestion.μ-Myc-pIRES2-EGFP,δ-FLAG-pIRES2-EGFP,κ-HA-pIRES2-EGFP recombinant plasmid was transfected into HEK293 using Lipofectamine2000 according to its instructions, respectively. The expression ofμ-Myc-pIRES2-EGFP,δ-FLAG-pIRES2-EGFP,κ-HA-pIRES2-EGFP in HEK293 was examined through immunofluorescence and western blot. The EGFP expression was observed through fluorescence microscopy using blue light, which showed the transfection efficiency of recombinant eukaryotic plasmid in HEK293 was high. Expression level ofδgene with FLAG epitope was detected in HEK293 afterδ-FLAG-pIRES2-EGFP plasmid transfection. We examined lysates from cells expressingδopioid receptors tagged with a FLAG epitope using FLAG primary antibody and corresponding secondary antibody tagged with horseradish peroxidase. The result was showed through enhanced chemiluminescence. We found that mostδreceptors exist as dimers of relative molecular mass (Mr) 140,000. The dimers are stable in 10% SDS buffer and 100℃water bath. At the same time, there were also being monomer regardless of glycosylation. Homotetramers and higher molecular mass oligomers were not observed. The result ofμ-Myc-pIRES2-EGFP andκ-HA-pIRES2-EGFP in HEK293 were similar to that ofδ-FLAG-pIRES2-EGFP in HEK293 using western blot. These results showed opioid receptors exist as homodimers in HEK293 using recombinant eukaryotic plasmid and the homodimers are stable in SDS detergents and 100℃water bath.We examined the ability ofμopioid receptors to heterodimerize withδopioid receptors by coexpressing Myc-taggedμreceptors with Flag-taggedδreceptors. The interaction betweenμandδopioid receptors was examined by immunoprecipitation experiments using HEK293 transfectedμ-Myc-pIRES2-EGFP andδ-FLAG-pIRES2-EGFP recombinant plasmid. Collecting the cells 48h after transient expression, immunoprecipitation and western blotting were carried out with lysates of whole cells using lysis buffer(Tris pH 7.4,50mmol/L, NaCl 120mmol/L, Triton-X100 1%, protease inhibitor cocktail(10 pg/ml leupeptin,10 pg/ml aprotinin,10 mM EDTA,1 mM EGTA,10 pg/ml bacitracin,1 mM pepstatin A and 0.5 mM phenylmethylsulphonyl fluoride)). We found that an antibody to the Myc-taggedμreceptor can co-precipitate Flag-taggedδreceptors from cells expressing both Myc-taggedμopioid receptor and Flag-taggedδopioid receptors, vice versa. The receptors could be co-precipitated only from cells cotransfectedμ-Myc-pIRES2-EGFP andδ-FLAG-pIRES2-EGFP, and not from a mixture of cells individually expressing the receptors. These results indicate that the heterodimerization betweenμandδopioid receptors is specific and selective. Coexppression ofμandδopioid receptor is necessary forμandδreceptor heterodimization.According to molecular structure topology map of ratδopioid receptor, we constructed prokaryotic expression plasmid of some domains ofδreceptor with pGEX2TK. These domains are the second intercellular loop, the third intercellular loop, the carboxyl terminal, the first transmembrane, the third transmenbrane, the fifth transmembrane, the sixth transmembrane, the full length ofδreceptor tagged with FLAG. These constructed prokaryotic expression plasmids are as follows:δⅡ-pGEX2TK,δⅢ-pGEX2TK,8C-pGEX2TK,δTM1-pGEX2TK,δTM3-pGEX2TK,δTM5-pGEX2TK,δTM6-pGEX2TK,δ-FLAG-pGEX2TK. ForδⅡ-pGEX2TK,δⅢ-pGEX2TK,δC-pGEX2TK, after transforming the recombinant prokaryotic expression plasmid into expression bacteria BL21 (DE3), we used IPTG to introduce the expression of GST fusion protein. Coomassie brilliant blue staining showed theδⅡ-pGEX2TK,δⅢ-pGEX2TK,δC-pGEX2TK successfully expressed GST fusion protein of target domain in BL21 (DE3). According to batch purification protocol of Glutathione Sepharose 4B, the GST-δⅡ,GST-δⅢ,GST-δC were collected and incubated with cell lysate form HEK293 transfected withμ-Myc-pIRES2-EGFP plasmid. The precipitation was analysed by western blot with Myc primary antibody. The results showed that the second intercellular loop, the third intercellular loop, the carboxyl terminal don't play an important role in interaction betweenμandδopioid receptor. Expression ofδTM1-pGEX2TK,δTM3-pGEX2TK,δTM5-pGEX2TK,δTM6-pGEX2TK,δ-FLAG-pGEX2TK failed in BL21 (DE3). We tried different concentration of IPTG (0.05,0.2,0.5,1.2mmol/L) different temperature (37,30, 25℃) different time (4,7,16h) different expression bacteria(BL21(DE3)plysS, Rosetta gami (DE3),Rosetta (DE3)),but still couldn't get the GST fusion protein. These results showed that it is difficult to express the membrane protein and hydrophobic transmembrane in prokaryotic expression bacteria.Conclusions1.μ-Myc-pIRES2-EGFP,δ-FLAG-pIRES2-EGFP,κ-HA-pIRES2-EGFP eukaryotic recombinant plasmid were constructed correctly and expressed in high level in HEK293 cells. These opioid receptors are mainly in the form of homodimer when expressed in HEK293 cells.2. It is difficult to express the membrane protein and hydrophobic transmembrane in prokaryotic expression system.3. Heterodimerization betweenμandδopioid receptor is specific and selective. Coexppression ofμandδopioid receptor is necessary forμandδreceptor heterodimization.4. The second intercellular loop, the third intercellular loop and the carboxyl terminal ofδopioid receptor don't play an important role in interaction betweenμandδopioid receptor.5. Our data indicated that the interaction betweenμandδopioid receptor may require other protein participation.