NR2B亚单位C末端调控NMDA受体运输和膜表达的分子机制

发布时间：2018-05-15 02:40

本文选题：NMDA受体 + 内质网滞留基序　；参考：《浙江大学》2008年博士论文

【摘要】： NMDA受体是一种谷氨酸门控的离子通道,与许多复杂的生理和病理机制有关,如突触的可塑性、长时程增强作用(LTP)、学习和记忆、兴奋性神经毒性、神经退行性变性疾病等。NMDA受体由NR1、NR2和NR3亚单位装配组成异聚体离子通道,在内质网组成具有不同生理学和药理学功能以及不同突触靶向的功能性NMDA受体通道。已知NR1亚单位是一个独立的基因家族,通过选择性剪接可产生8个剪接变体;NR2共有4个相关的基因,NR2A、NR2B、NR2C和NR2D:NR3则有2个基因,分别为NR3A和NR3B。目前认为,NMDA受体是分别由两个结合甘氨酸的NR1和两个结合谷氨酸的NR2亚单位组成的异四聚体。因此,NR1/NR2异源二聚体就组成了NMDA受体的基本功能单位。先前的大量研究表明,NMDA受体的不同亚单位单独表达都被滞留在内质网内,只有当NR1与NR2亚单位共同装配才能从内质网输出并表达到细胞膜表面,但其作用机制仍不清楚。近几年来,研究发现NR1亚单位存在一些重要的功能区和调节位点:例如,NR1-1亚单位的C1区存在一个RRR内质网滞留基序(ERretention motif),使NR1亚单位被滞留在内质网;NR1-4a存在一个共感的PDZ结合区,可以掩盖内质网滞留基序;PKC活化可以帮助NR1-1a表达于细胞膜表面等等。然而,对于NR2B亚单位的内质网滞留机制却仍然知之甚少。与NR1不同的是NR2B亚单位的C末端很长,我们先前的研究已经发现,缺失NR2B亚单位C末端不同区域均未能使得NR2亚单位从内质网输出并获得表面表达,这表明NR2亚单位的内质网滞留机制要比NR1复杂得多。已知NR1-4a单独表达能到达细胞膜表面,为了明确是否是NR2B亚单位C末端决定了它的自身滞留,我们将NR1-4a的C末端替换到NR2B的亚单位C末端。有趣的是,我们发现这样的一个NR2B_(NR4aC)的嵌合体获得了细胞膜表面表达,这个结果表明NR2B亚单位C末端决定了NR2B亚单位的细胞膜表达。但是关于NR2B亚单位C末端如何调控NR2B亚单位的内质网滞留作用仍然不清楚,我们推测并不是由单一位点决定,很可能是通过多个位点协同作用的结果,这还需要进一步的研究证明。另外,关于NR2亚单位单独表达是否能形成功能性受体的研究一直是这个领域的一个热点,目前的主流观点推测NR1是NMDA受体得必需组分,NR2亚单位单独并不能形成有功能得离子通道。基于NR2B_(NR4ac)能单独到达细胞膜表面,我们运用全细胞电生理的方法检测了NR2B_(NR4ac)嵌合体是否能形成有功能的受体通道。电生理结果没有记录到明确的NMDA受体样电流。这个结果提示单独表达NR2亚单位并不能形成有功能的受体通道,只有与NR1共装配才能形成有功能的通道。其次,已知NR1-1a可以通过与NR2装配克服其内质网滞留作用并被输送到细胞膜表面,但其作用机制并不清楚。我们先前的研究试图在NR2上找到克服NR1a内质网滞留基序的功能区,做了各种缺失突变体,但均未能找到特殊的功能区。在本研究中,我们构建了NR2B各种C末端截短质粒,结果发现除了2BA2不能与NR1-1a获得细胞膜表面表达之外,其它的缺失体都能与NR1-1a共表达于细胞膜表面。进一步用全细胞膜片钳技术来记录转染细胞的NMDA受体电流,结果与表面染色结果一致。这些结果表明,NR2B的C末端氨基酸长度对克服NR1-1a的内质网滞留有重要作用,换句话说,NR2亚单位C末端保留三个氨基酸长度对于克服NR1-1a的内质网滞留基序作用是必需的。为了验证这个结果,我们获得了一个NR1-1a-AAA突变体,只把NR1-1a的RRR滞留基序突变成了AAA。然后分别把2B△2与NR1-1a-AAA突变体共转染到HEK293细胞,表面染色结果显示它们能获得细胞膜表达。接下来我们想知道的一个问题是这个氨基酸序列是否具有特异性呢?为了回答这个问题,我们构建了NR2B△5_(AAAAA)突变体,将它与NR1-1a共转染到HEK293细胞后,表面染色和电生理结果都表明它们形成的复合物仍然能够到达细胞膜表面并形成有功能的受体。这个结果表明NR2B第四跨膜区后三个氨基酸长度并没有序列特异性。基于上述结果,本研究第一次发现NR2亚单位C末端保留三个氨基酸长度对于克服NR1-1a的内质网滞留基序是必需的。这种调控机制很可能是通过引发NR1亚单位C末端自身变构的改变,从而掩盖了NR1-1a C末端的内质网滞留基序,使得NR2△3/NR1-1a获得细胞膜表面表达。本研究揭示了一种新的NMDA受体亚单位装配和运输调控机制。另外,本研究运用FRET技术来观察NR2B亚单位的C末端是否影响NMDA受体的装配。将构建好的YFP-2B△2,CFP-2B△2质粒共转染到HEK293细胞,发现它们仍然能够发生FRET,这表明YFP-2B△2与CFP-2B△2能形成复合物。同时,分别将YFP-2B△2与CFP-NR2B或者CFP-NR1-1a共转染到HEK 293细胞,结果发现它们也能发生FRET。这些结果说明缺失了C末端的NR2B亚单位仍然能够与NR1装配形成NMDA受体复合物,提示NR2B的C末端并不是NMDA受体装配的决定位点。最后,在培养的海马神经元中分别单独转染GFP-NR2A,GFP-NR2B,2A△5,2A△3,2A△2,2B△3和2B△2,活细胞表面染色观察它们在神经元的细胞膜表达情况。结果显示2A△5,2A△3,2A△2单独表达到神经元均能到达细胞膜表面,尽管与野生型相比有显著降低。有趣的是,2B△2单独表达在神经元中并不能获得表面表达,但是将NR1-4a与2B△2共转染到神经元中则能够观察到2B△2到达了细胞膜表面,虽然跟野生型相比膜表面表达数目仍然有明显下降。众所周知,在神经元中存在各种内源性的NR1剪接变体,如果NR2能与这些不同的NR1变体装配的话,那么2B△2应该能与NR1-4亚单位装配并到达细胞膜表面,但结果发现2B△2不能到达细胞膜表面。这个结果暗示了一种新的现象,即不同的NR2亚单位并不是随机的与各种NR1变体装配形成NMDA受体,很可能与NR2A和NR2B装配的NR1是不同的变体。综上,本研究主要结论如下:1)发现NR2B_(NR4aC)的嵌合体能获得细胞膜表面表达,电生理结果提示单独表达NR2亚单位不能形成功能性离子通道;2)本研究首次发现NR2亚单位C末端保留三个氨基酸长度对于克服NR1-1a的内质网滞留基序是必需的,揭示了一种新的NMDA受体亚单位装配和运输调控机制。
[Abstract]:NMDA receptor is a glutamate gated ion channel, which is related to many complex physiological and pathological mechanisms, such as synaptic plasticity, long term enhancement (LTP), learning and memory, excitatory neurotoxicity, neurodegenerative disease, etc..NMDA receptors are assembled by NR1, NR2 and NR3 subunits to form an isomer ion channel in the endoplasmic reticulum group. As a functional NMDA receptor channel with different physiological and pharmacological functions and different synaptic targets, the known NR1 subunit is an independent gene family, and 8 splice variants can be produced by selective splicing; NR2 has 4 related genes, NR2A, NR2B, NR2C and NR2D:NR3 have 2 genes, NR3A and NR3B. are currently considered, NMD, respectively, NMD. The A receptor is an hetero four polymer consisting of two binding glycine NR1 and two NR2 subunits that bind glutamate. Therefore, NR1/NR2 heterologous two polymers constitute the basic functional units of the NMDA receptor. A large number of previous studies showed that the different subunits of the NMDA receptor were isolated in the endoplasmic reticulum alone, only when NR1 and NR2 subunits were subdivided. Unit assembly can be assembled from the endoplasmic reticulum to the surface of the cell membrane, but the mechanism of its action is still unclear. In recent years, some important functional areas and regulatory sites have been found in NR1 subunits, for example, a RRR endoplasmic reticulum retention base (ERretention motif) exists in the C1 region of the subunit of NR1-1 and the NR1 subunit is detained. NR1-4a has a shared PDZ binding area that can cover the retention of the endoplasmic reticulum; PKC activation can help NR1-1a to express on the surface of the cell membrane. However, little is known about the mechanism of the endoplasmic reticulum retention in NR2B subunits. Unlike NR1, the C end of the NR2B subunit is long, and our previous study has found the deletion. The C terminal of the subunit of the NR2B subunit failed to make the NR2 subunit output from the endoplasmic reticulum and obtain the surface expression. This indicates that the endoplasmic reticulum retention mechanism of the NR2 subunit is much more complex than that of NR1. The known NR1-4a expression can reach the surface of the cell membrane. In order to determine whether it is the C terminal of the NR2B subunit, it determines its self retention. We will NR1-4a The C terminal was replaced by the subunit C terminal of NR2B. Interestingly, we found that such a NR2B_ (NR4aC) chimeras obtained the expression of the cell membrane surface. This result indicates that the C terminal of the NR2B subunit determines the cell membrane expression of the subunit of NR2B, but how the end of the C end of NR2B subunit regulates the retention of the endoplasmic reticulum of NR2B subunit. It is still not clear that we speculate that it is not determined by a single locus, it is likely to be the result of synergistic effects of multiple sites, and this needs further research. In addition, the study of whether NR2 subunits can form functional receptors alone has been a hot spot in this field, and the current mainstream view is that NR1 is NMDA. The receptor is essential component, NR2 subunit alone does not form a functional ion channel. Based on the ability of NR2B_ (NR4ac) to reach the surface of the cell membrane alone, we use the whole cell electrophysiological method to detect whether the NR2B_ (NR4ac) chimeras can form a functional receptor channel. Electrophysiological results do not record a clear NMDA receptor like current. This result suggests that NR2 subunits alone can not form functional receptor channels. Only functional assembly can be combined with NR1 to form functional channels.
Second, it is known that NR1-1a can overcome its endoplasmic reticulum retention by assembly with NR2 and be transported to the surface of the cell membrane, but its mechanism is not clear. Our previous study tried to find a functional area to overcome the retention of the NR1a endoplasmic reticulum on NR2, and did various missing mutants, but failed to find special functional areas. In addition, we constructed various C terminal truncated plasmids of NR2B. It was found that other missing bodies could be co expressed with NR1-1a on the surface of the cell membrane except that 2BA2 could not obtain the expression of cell membrane surface with NR1-1a, and the whole cell patch clamp technique was used to record the body current of NMDA in the transfected cells. The results were in agreement with the results of surface staining. The results show that the C terminal amino acid length of NR2B has an important effect on overcoming the endoplasmic reticulum retention of NR1-1a. In other words, the retention of three amino acid lengths at the C terminal of NR2 subunit is necessary for overcoming NR1-1a's endoplasmic reticulum retention basis. In order to verify this result, we have obtained a NR1-1a-AAA mutant, only RRR hysteresis of NR1-1a. The retention of the sequence turned into AAA. and then co transfected 2B delta 2 with NR1-1a-AAA mutants to HEK293 cells, and the surface staining results showed that they were able to obtain cell membrane expression. One problem we wanted to know was whether the amino acid sequence was specific? To answer this question, we constructed the NR2B Delta 5_ (AAAAA) mutation. After CO transfection of it and NR1-1a into HEK293 cells, surface staining and electrophysiological results showed that their formed complexes could still reach the surface of the cell membrane and form a functional receptor. The results showed that the length of the three amino acids after the fourth transmembrane region of NR2B was not sequence specific. Based on the above results, the first time of this study was made. The retention of three amino acid lengths at the C terminal of the present NR2 subunit is necessary for overcoming the endoplasmic reticulum retention motif of NR1-1a. This regulation mechanism is likely to conceal the retention of the endoplasmic reticulum at the NR1-1a C terminal by initiating a change in the self allosteric structure of the C terminal of the NR1 subunit, which makes NR2 Delta 3/NR1-1a obtain the surface expression of the cell membrane. A new mechanism of NMDA receptor subunit assembly and transport is revealed.
In addition, this study uses FRET technology to observe whether the C terminal of NR2B subunit affects the assembly of NMDA receptors. The constructed YFP-2B delta 2, CFP-2B delta 2 plasmids are co transfected to HEK293 cells, and they are still able to occur FRET, which indicates that YFP-2B delta 2 and CFP-2B delta 2 can form complex. -1a was co transfected to HEK 293 cells, and it was found that they could also produce FRET.. These results suggest that the NR2B subunit missing the C terminal can still be assembled with NR1 to form a NMDA receptor complex, suggesting that the C terminal of NR2B is not a determining site for the assembly of NMDA receptors.
Finally, GFP-NR2A, GFP-NR2B, 2A Delta, 3,2A Delta 2,2B delta 3 and 2B delta 2 were transfected separately in the cultured hippocampal neurons. The expression of them in the cell membrane of the neurons was observed by living cell surface staining. The results showed that 2A Delta 5,2A Delta 3,2A delta 2 alone could reach the surface of the cell membrane, although it was significant compared with the wild type. Interestingly, it is interesting that 2B delta 2 can not be expressed in neurons alone, but NR1-4a and 2B delta 2 co transfected into neurons can observe the arrival of 2B delta 2 on the surface of the cell membrane, although there is a significant decrease in the number of membrane surface expressions compared with the wild type. It is well known that there are various endogenous NR in the neurons. 1 splice variants, if NR2 can be assembled with these different NR1 variants, then 2B delta 2 should be able to assemble and reach the surface of the cell membrane with NR1-4 subunits, but the result is that 2B delta 2 cannot reach the surface of the cell membrane. The result suggests a new phenomenon that different NR2 subunits are not randomly assembled to form NMD with various NR1 variants. The A receptor is probably different from the NR1 assembled by NR2A and NR2B.
To sum up, the main conclusions of this study are as follows: 1) it is found that the chimeras of NR2B_ (NR4aC) can be expressed on the surface of the cell membrane. Electrophysiological results suggest that NR2 subunits can not form functional ion channels alone. 2) this study first found that the length of three amino acids in the C terminal of NR2 subunit is necessary to overcome the retention of the endoplasmic reticulum of NR1-1a. It reveals a new mechanism of NMDA receptor subunit assembly and transport regulation.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R33

【引证文献】