The Preliminary Study Of Interactions Between I₁-imidazoline Receptor And μ-opioid Receptor

Imidazoline receptors, were found in 1980's, which were named because ofshowing high affinity to compounds containing the structure of"imidazoline rings".Currently, it is generally accepted that three subtypes I₁, I₂ and I₃ exist, according totheir differences in affinity to selective ligands, tissue distributions, celluar functionsand so on. In 2000, a gene encoding an I₁ imidazoline receptor candidate protein,named imidazoline receptor antisera-selected protein (IRAS), was cloned from humanhippocampusλgt11 cDNA library by professor Piletz. Subsequently, several lines ofevidences support the identity of native I₁-imidazoline receptor and cloned IRAS.Agmatine that is synthesized following decarboxylation of L-arginine is one of theendogenous ligands as well as non-specific agonist for imidazoline receptors. In thepast decade, more and more accumulated results reported by our laboratory indicatedthat agmatine has obvious actions on opioid functions. Exogenous agmatine couldenhance morphine analgesia, attenuate morphine tolerance, physical and psychologicaldependence. Endogenous agmatine has similar functions. Moreover, down-regulatingthe expression of IRAS in CHO cells coexpressing MOR and IRAS or the endogenousI₁R in hippocampal neurons and mouses by using non-specific antagonist and RNAinterference technology demonstrated that the target of agmatine regulating morphinedependence was related to I₁R. Therefore, we presumed that I₁R may involve inmodulation of functions of opioids. However, the molecular mechanisms of I₁Rinvolved in agmatine inhibiting opioid tolerance and dependence remain unclear. Ourprevious study showed that there was significant difference in desensitization of MORin CHO-MOR and CHO-MOR/IRAS. However, the reasons for the difference remainuncleared. For distribution of I₁R overlaps that of MOR in brain regions. By analysingthe structure of I₁R, it contains key domains for interaction with other proteins, whichsuggesting IRAS may modulate functions of MOR by interaction with MOR. In therecent years, accumulated evidences supported that opioid receptors could form homo or heterodimers/oligomers with members belong to the same family or differentfamilies. Dimers/oligomers may be a new kind of modulation mechanism, whichinhibit or reinforce the phosphorylation, desensitization of opioid receptors anddimers/oligomers between/among opioid receptors as well as other G-protein coupledreceptors. A series of studies showed that some non-opioid receptors as dopamine D1receptor orα₂ adrenergic receptor (α₂AR) could form heterodimers, which modulatesopioid dependence and tolerance. Therefore, we presumed whether I₁R could modulatethe functions of MOR through dimerization/oligomerization with it. Based on theabove analysis, we carried out the following studies.Firstly, in the model of HEK293 cells that were transiently transfected plamids ofHA/Flag-MOR or/and Myc-IRAS, we employed immunocytochemistry to observe thesubcellular location of MOR and IRAS. The results of laser confocal pictures showedthe co-localization of MOR and IRAS when they co-expressed. There existedco-localization of both proteins especially in the membrane. In the cerebral cortex andDG of hippocampus, we found endogenous I₁R and MOR could also co-localize in theneuron. Co-localization of subcellular distribution suggested possibility of interactionsbetween IRAS and MOR. Subsequently, different methods were employed fordemonstration the formation of heterodimers of IRAS and MOR. The classic methodfor studying protein interactions coimmunoprecipitation was used. The MOR could beimmunoprecipitated by IRAS, and IRAS was also immunoprecipitated by MOR in theimmunoprecipitation and reciprocal immunoprecipitation experiments. Neither MORnor IRAS interacted with uncorrelated protein GFP. We further found MOR couldco-immunoprecipitation with IRAS in CHO-MOR/IRAS, in which both proteinsexpressed at lower level than in HEK293 transfected transiently. These resultspreliminarily indicated that IRAS could interact with MOR in vitro. Meanwhile, highconcentration reductant DTT (100 mM) did not interrupt the interactions, indicatingthat the interactions may be not mediated by disulfide bond. Agonists (DAMGO oragmatine) treatment had no obvious effect on the level of heterodimerization betweenboth proteins. For further confirming the results of co-immunoprecipitation, newlyemerging approach fluorescence resonance energy transfer was also employed. Thefindings from fluorescence resonance energy transfer showed that there existedinteractions between IRAS and MOR.Whether the interactions between IRAS and MOR would affect the signaltransduction and the adaptation of MOR after agonist treated. Therefore, the desensitization of MOR was investigated in CHO-MOR and CHO-MOR/IRAS. Firstly,we found that there is no significant difference in the ability of DAMGO inhibitingadenylate cyclase (AC) by activating MOR between CHO-MOR andCHO-MOR/IRAS, indicating interactions of IRAS and MOR had no effect on cAMP,the important second messenger of MOR signal transduction. Nextly, desensitizationof MOR was studied to reflect the adaptation of MOR. We pretreated the two lines ofcells simultaneously with DAMGO for different times (0, 7.5 min, 15 min, 30 min, 45min, 1 h, 2 h, 3h, 4h) to investigate whether there were differences in desensitization ofMOR in CHO-MOR and CHO-MOR/IRAS. We found that CHO-MOR cells werepretreated with DAMGO in 0-4 h, MOR showed time-dependent desensitization.When DAMGO pretreated for 4 h, the rate of MOR desensitization was around 40%.However, MOR in CHO-MOR/IRAS showed fast desensitization and the trend ofresensitization subsequently in 0-1 h. With the prolonged pretreatment(1-4 h), the rateof desensitization grew slower and the level of desensitization showed significantdifferences compared with CHO-MOR. When DAMGO pretreated for 4 h, the rate ofMOR desensitization was around 20% in CHO-MOR/IRAS. Subsequently, we furtherstudied the influence of the interaction between MOR and IRAS on resensitization ofdesensitized MOR. DAMGO pretreated both two cell lines for 4 h and removedagonist, then kept cellls in incubator for diffent intermissions of 10 min, 30 min, and 1h. The result showed that MOR in both CHO-MOR and CHO-MOR/IRAS displayedthe trend of resensitization during. After 1 h, the rate of MOR resensitization inCHO-MOR/IRAS was around 13%, which higher than 9.7% in CHO-MOR. In view ofreceptor resensitization correlating with recycling to membrane, we presumed that theinteractions of IRAS with MOR could lead to accelerating MOR resensitization byinflucing the trafficking of MOR.In conclusion, Present study revealed the existence of interactions of IRAS withMOR at receptor level through heterodimerization for the first time, which may be notmediated by disulfide bond and not agonist-dependent. In addition, IRAS couldaccelerate MOR resensitization, which may be related to the interaction with MOR.Desensitization of receptors is one of mechanisms for tolerance to opioids. Reducingdesensitization meansthe possibility of abating opioids tolerance and dependence. Theresults above indicate I₁R could attenuate the extent of MOR desensitization byinteractions with MOR, suggesting I₁R is a key molecular to modulate opioid functions.Meanwhile, our study provide new theoretical basis for developing the novel drugs for treating addiction and pain.