| The kappa opioid receptor (KOR) accompanied with its exclusive endogenous ligands,dynorphins, are broadly expressed across the central nervous system, besides the classical G-protein coupled pathway mediating analgesia, non G-protein dependent pathway, especially the P-arrestin dependent pathway, often invokes the side-effects mediated by KOR. While striatum is well known for its modulation in the forming of compulsive or habit-like behaviors,such as drug abuse or addiction, and furthermore a pronounced elevated KOR/dynorphin system can be even taken as a hallmark of the drug addiction, and furthermore the promoted KOR/dynorphin system may contribute much to the drug abuse and addiction through special biased ?-arrestin2-dependent pathway. Therefore,it's extremely urgent to find out the root cause for the biased ?-arrestin dependent pathway according to the activation of KOR in striatum.While at spinal levevl, 6'-GNTI, a category of KOR-DOR selective agonist, could be potent analgesic,but no such effect could be observed when intraventricular administration carried out, combinding the the universality of the existence of GPCR heterodimers, and novel structural pharmacological characters distinct from that of individual ones, all these highy suggested the possibility of different expression forms of KOR existing in brain whose function might distinct from that of KOR monomers or KOR-DOR heterodimers owing to the activation of the non-classical pathway. Although NT/NTSRl system has been widely studied for its functions overlapping with that of dynorphin/KOR system, little was known about the direction between KOR and NTSR1.And fortunately, preliminary experiments using laser confocal microscopy have already verified the co-localization in cultured striatal neurons, and additional Proximity Ligation Assay (PLA) have already confirmed the endogenously distributed KOR-NTSR1 heterodimers, which provided with a solid structural substrate for our prediction.Thus, as there was really heterodimers composed of KOR and NTSR1 in primary cultured striatal neurons, the current study was attempting to obtain further confirmation of the heterodimerization effects between KOR and NTSR1, and to verify the key residue involved in the formation of KOR-NTSR1 heterodimers, and furthermore, to reveal the possible alteration of signal transduction owing to the interaction between KOR and NTSR1.(1) We firstly constructed various expression vectors of KOR and NTSR1, and then HEK293 cell models expressing KOR and/or NTSR1 were obtained by transiently or stable transfection experiments. western blotting and ELISA experiments succeeded in detecting KOR expressing in HEK293 KOR cell lines and HEK293 KOR/NTSR1 cell lines, and NTSR1 expressing in HEK293-NTSR1 cell lines and HEK293 KOR/NTSR1 cell lines as well also, and the expression level of KOR or NTSR1 was not significantly affected by co-expression, and confocal microscopy assay represented that KOR and NTSR1 were evenly distributed at the plasma membrane, and an undoubtful co-localization in this area in the KOR/NTSR1 co-expressing cells. All the evidences above indicated that the cell models was being correctly established and could be used in subsequent research.(2) 184 and 196 residue mutants (point mutants of leucine to alanine in/around TM4) of KOR were obtained by SOE-PCR,and then HEK293 cell models expressing KOR184/196 and/or NTSR1 were obtained by transiently or stable transfection experiments. Confocal microscopy, western blotting and ELISA were also carried out to investigate the expression of mutant KORs transfected HEK293 cells. The data suggested that KOR184 or KOR196 mutants showed little difference from the expression level compared to the wt KOR, and as also is the case with the expression of KOR184 or KOR196 in co-transfected HEK293 with NTSR1 compared to the wt KOR.(3) Direct interaction between KORWT and NTSR1 was confirmed by BRET, FRET and CO-IP assays, while the additional data presented in our research suggested that KOR196 but not KOR184 failed to form the receptor heterodimers with NTSR1 in co-expressed HEK293 cells, indicating that leucine 196 residue of KOR was required for KOR-NTSR1 heterodimerization. Moreover,KOR196 and NTSR1 co-transfected HEK293 cells could be used as a negative control in discussing the possible signal integration or conformational modulation mediated by the KOR-NTSR1 heterodimer(4) The intracellular cAMP level was investigated to explore the potential effects of KOR-NTSR1 heterodimerization on KOR-activated signaling transduction. In HEK293 cells stably expressing KORs (KORWT or KOR184,or KOR196),forskolin-stimulated adenylate cyclase activity was significantly inhibited by dynophin A(1-13) in a dose-dependent manner(KORWT: maximal inhibition = 66.5%, KOR184: maximal inhibition = 70%, KOR196: maximal inhibition = 63.9%). A similar dose-dependent inhibition induced by dynophin A(1-13) like above was also found in HEK293 KOR196/NTSR1 cells (maximal inhibition = 59%), while merely a little decrease of the intracellular cAMP level occured in HEK293 KORWT/NTSR1 or KOR184/NTSR1 cells when treated with dynophin A(1-13) only ( KORWT: maximal inhibition = 14.7%, KOR184: maximal inhibition = 14.9%), which highly indicated that dimerizing with NTSR1 almost eliminated the ability of KORWT (or KOR184) in inhibiting adenylate cyclase activity in KORWT/NTSR1 (or KOR184/NTSRI) cells.(5) BRET assay revealing the interaction between KORs and Gai2 protein was subsequently carried out to confirm the validity of above cAMP results. When treated with dynorphin A(1-13), the BRET ratio in cells expressing Rluc-Gai2 and Venus-tagged KORWT(or KOR184) underwent an abrupt increase with a peak at 5 min, which was abbreviated in cells co-expressed with untagged NTSR1. While such variation did not occur in Rluc-Gai2,KOR196-Venus and NTSR1 expressed cells where NTSR1 couldn't dimerize with KOR196.(6) To explore the possible variation in the (3-arrestin recruiting ability of KOR associated with the formation of NTSR1-KOR heterodimer, BRET1 and eBRET were carried out in ?-arrestinl/2-Rluc and KORs-Venus transfected HEK293 cells with or without the expression of un-tagged NTSR1. The BRET ratio between ?-arrestinl/2-Rluc and KORWT-Venus (or KOR184-Venus) underwent a much more robust increase with the co-expression of un-tagged NTSR1 compared with that of cells with only ?-arrestinl/2-Rluc and KORWT-Venus (or KOR184-Venus) were expressed, and the duration of the BRET ratio increase was extend to nearly 2 h for the former, and only 30 min for the latter.(7) Western blotting was used to investigate the activation of ERK1/2 in KORs and/or NTSR1 expressing HEK293 cells. In HEK293 cells expressing KORWT (or KOR184) and NTSR1, besides the promptly ERK activation increase at 5 min after dynorphin A(1-13)treatment, a sustained increase was also found, and maintained significant high levels at 30 min time point,compared with that of KORWT (or KOR184)singly expressed cells. While such sustained ERK1/2 phosphorylation really was not acquired in KOR196/NTSR1 cells.(8) To study the possible roles of P-arrestin 1/2 in KORWT-NTSR1 heterodimer mediated ERK1/2 activation in detail, ?-arrestinl/2 shRNA was used in HEK293 cells expressing KORWT or/and NTSR1. In KORWT/NTSR1 co-expressed HEK293 cells, the intense sustained ERK1/2 activation at 30 min induced by dynorphin A(1-13) was nearly abolished by the treatment of ?-arrestin2 shRNA, and even the early rapid ERK1/2 activation at 5 min was also attenuated compared with the control shRNA treated cells. While in PTX experiments, the early rapid ERK 1/2 phosphorylation at 5 min in KORWT singly expressed cells induced by dynorphin A (1-13) was almost abolished by PTX, indicating a G protein-dependent pathway involved in KORWT signaling. While similar result was not obtained in KORWT and NTSR1 co-expressed HEK293 cells, there was only a minor reduction (40%) of early rapid ERK1/2 phosphorylation induced by dynorphin A(1-13) appeared after PTX treatment, but the sustained ERK1/2 phosphorylation was not affected at all especially at 30 min, suggesting G protein-dependent signaling of KORWT should be attenuated after it being dimerized with NTSR1, while non-G protein-dependent signaling increased, which strengthened the discovery in P-arrestins shRNA experiments.(9) The reduced ability of KORWT in inhibiting of cAMP, activating of Gi2 and enhanced recruitment of P-arrestins in KORWT/NTSR1 co-expressing cells in response to dynorphin A(1-13) were almost fully set back to to that of KORWT singly expressed cells by the co-incubation of NT(8-13), whiche highly predicted that there should be something different in the proximity interaction between two protomers due to occupation of NTSR1.(10) BRET assays were carried out to determine the possible changes of KORWT-NTSRI heterodimerization induced by ligands. NT(8-13) induced a dose-dependent decrease in BRET signals observed in HEK293 cells co-transfected with NTSR1-Rluc and KORWT-Venus, similar changes was found when co-treated with various concentration of NT(8-13) plus dynorphin A(1-13), but no such dose-dependent manner was detected when treated only with dynorphin A(1-13) at various concentration. PLA experiment was used to quantify the heterodimers before and after the stimulation of agonists. In cells stably transiently transfected with KORWT and NTSR1, the positive PLA products were significantly reduced after incubation with NT(8-13) for 30 min, and remained almost unchanged when confronted with dynorphin A(1-13), when the cells were treated with dynorphin A(1-13) and NT(8-13) simultaneously, while the positive PLA products still underwent significantly reduction similarly with NT(8-13) incubated cells. Combined results in BRET assays and PLA experiments, we could infer that KORWT and NTSR1 did form heterodimers, but these two partners would probably experience conformational changes within the dimer or undergo dissociation in the presense of NT(8-13), regardless of the existence of dynorphin A(1-13).We finally investigated the impact that NT(8-13) had on sustained ERK activation induced by dynorphin A(1-13) in HEK293 KORWT/NTSR1 cells. While sustained ERK1/2 activation at 30 min was undetected in the KORWT/NTSRI cells incubated with dynorphin A(1-13) in the presence of NT(8-13) and further dose-dependent assay was then carried out to verify the above result, in which NT(8-13) exhibited a dose-dependent surpressing ability in dynorphin A(1-13) mediated ERK 1/2 activation at 30 min. These evidences indicated that NTSR1 acting could act as a switch for G protein or non G protein (?-arrestin2) dependent signaling pathway mediated by KOR.In the current study, we identified the KOR-NTSR1 heterodimers for the first time not only in transfected HEK293 cells, but also in primary cultured striatal neurons. And we also found that the abilities of KORWT in inhibiting of cAMP and activating of Gi2 were much reduced, recruitment of p-arrestins enhanced and even a sustained ERK1/2 activation could be obtained in KORWT/NTSR1 co-expressing cells in response to dynorphin A (1-13),all these suggested dimerizing with NTSR1 brings a switch to KOR mediated signaling transduction,from G protein-dependent manner to ?-arrestin2-denpendent pathway, which fully verified our prediction of NTSR1 being parterner of KOR in striatum, and provided solid theoretical basis for the biased ?-arrestin dependent pathway according to the activation of KOR in striatum, which inturn contributed much in elucidating the mechanism of drug abuse and addiction. And furthermore, NT(8-13) in this research was found to be able to modify the KOR-NTSR1 heterodimer complex in conformation or in association aspects, thus the activation of NTSR1 drawed ?-arrestin2 dependent signaling back to major G protein coupling pathway mediated by KOR. Therefore, this result not only provided an evidence for the NT/NTSR1 system's participating the modulation of pain and its antipsychotic profiles,but also reminded us of the impairment of KOR-NTSR1 complex could be a new way to treat drug addiction, in which the simultaneously elevated dynorphin/KOR system could be brought into full play for its bona fide effects. |
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