MOLECULAR CHARACTERIZATION OF GLUTAMATE RECEPTOR EXPRESSION IN BRAIN

谷氨酸受体(GluRs)与简单形式的 学习和记忆是几种精神活性物质的作用场所 毒品。最初有人提出谷氨酸能激活三种 不同的药理作用部位称为红藻氨酸、AMPA和NMDA 然而，最近的发现表明，海人藻酸盐和AMPA受体可以 争夺一个共同的受体位置。一个由四个基因编码的家族 AMPA/海人藻酸受体，称为Glur1-4(或G]ur A-D)，是最近发现的 描述。选择性剪接产生每个Glur的两种亚型 在转基因细胞中表达的亚单位，命名为FliP和Flop 形成表现出不同电生理特性的通道。 Glur-1亚单位的序列被用来克隆一个 起源于第三种类型的Glur4转录本；该cdna被称为 Glur-4c是因为它编码一种具有不同C末端的蛋白质。我们有 表明Glur-4c mRNAs来自另一种类型的差异RNA 加工；替代的C-末端可能不仅限于GluR-4，还 存在于来自其他GluR基因的蛋白质上。使用以下工具获得的结果 非洲爪哇卵母细胞表达系统与双电极联用 电压钳，证明Glur-4c基因编码的转录本 在注射的卵母细胞中形成功能性的同源低聚通道，这些通道在 对红藻氨酸、AMPA和谷氨酸的反应。世界银行的EC50 红藻氨酸的响应为44微米，由红藻氨酸引起的内向电流 通过与奎斯奎宁或AMPA共用大大减少了施用量， 提示这些激动剂在Glur-4c受体上有一个共同的结合部位。 对来源于Glur-4基因的转录本进行Northern分析 Blotts，并通过原位杂交，使用特定的寡核苷酸探针 不同的剪接变体。6.2、4.2和3.0 KB的三个频段 当与建议中的序列对应的探针时观察到 利用受体的N端胞外区进行杂交 含有小脑或培养的颗粒细胞的RNA的印迹。在……里面 相反，在含有RNA的印迹中没有观察到较小的转录本 来自小脑星形胶质细胞。与特异性的探针进行杂交 Flip、Flop和C-末端结构域仅与这两个结构域相关 较高分子量条带。原位杂交结果显示， Glur-4转录本在不同的小脑细胞中优先积聚 类型及其表达在发育过程中受到不同的调节。

Glutamate receptors (GluRs) have been implicated in simple forms of learning and memory and are the site of action of several psychoactive drugs. Initially it had been proposed that glutamate activates three distinct pharmacologically sites known as the kainate, AMPA and NMDA receptors, however, recent findings demonstrate that kainate and AMPA can compete for a common receptor site. A family of four genes coding for AMPA/Kainate receptors, called GluR 1-4 (or G]uR A-D), has been recently described. Alternative splicing generates two isoforms of each GluR subunit, designated flip and flop, that when expressed in transfected cells form channels that exhibit different electrophysiological properties. Sequences from the GluR-1 subunit cDNA were used to clone a cDNA originating from a third type of GluR 4 transcript; the cDNA was called GluR-4c because it codes for a protein with a distinct C-terminus. We have shown that GluR-4c mRNAs arise from yet another type of differential RNA processing; alternate C-termini may not be restricted to GluR-4 but also present on proteins derived from other GluR genes. Results obtained using a Xenopus oocyte expression system, in conjunction with two-electrode voltage clamp, demonstrate that the transcripts coded by the GluR-4c cDNA form functional homo-oligomeric channels in injected oocytes that open in response to kainate, AMPA and glutamate application. The EC50 of the kainate response is 44 microM and the inward currents elicited by kainate application are greatly reduced by coapplication of quisqualate or AMPA, suggesting a common binding site on GluR-4c receptors for these agonists. Transcripts originating from the GluR-4 gene were analyzed on Northern blots, and by in situ hybridization, using oligonucleotide probes specific to the different splice variants. Three bands of 6.2,4.2, and 3.0 Kb were observed when probes corresponding to sequences present in the proposed N-terminal extracellular domain of the receptor were used to hybridize blots containing RNA from cerebellum or cultured granule cells. In contrast, the smaller transcript was not observed in blots containing RNA from cerebellar astrocytes. Hybridization with probes specific to the flip, flop and C-terminal domains was associated exclusively to the two higher molecular weight bands. In situ hybridization revealed that the GluR-4 transcripts preferentially accumulate in different cerebellar cell types and their expression is differentially regulated during development.