Expression of endogenous NMDAR1 transcripts without receptor protein suggests post-transcriptional control in PC12 cells.
Expression of endogenous NMDAR1 transcripts without receptor protein suggests post-transcriptional control in PC12 cells.
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DOI：
10.1016/s0021-9258(18)41528-1
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发表时间：
1993-10
期刊：
影响因子：
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通讯作者：
N. Sucher;Nils Brose;D. Deitcher;M. Awobuluyi;Gregory Gasic;H. Bading;Constance L. Cepko;Michael E. Greenberg;Reinhard Jahn;Stephen F. Heinemann
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文献类型：
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作者：
N. Sucher;Nils Brose;D. Deitcher;M. Awobuluyi;Gregory Gasic;H. Bading;Constance L. Cepko;Michael E. Greenberg;Reinhard Jahn;Stephen F. Heinemann
用北方杂交法检测神经生长因子分化和未分化的大鼠嗜铬细胞瘤（PC 12）细胞中N-甲基-D-天冬氨酸受体NMDAR 1亚单位的RNA表达。在PC 12细胞中的NMDA受体1型（NMDAR 1）的信息在大小上与海马神经元中表达的信息相似。通过聚合酶链反应用NMDAR 1编码区侧翼的引物扩增的PC 12细胞cDNA对应于NMDAR 1剪接变体NMDA受体1型同种型C（NMDAR 1C）。采用钙离子显像和膜片钳技术，在PC 12细胞中未发现有功能的NMDA门控离子通道。为了研究PC 12细胞中是否存在NMDAR 1蛋白，开发了针对NMDAR 1的单克隆抗体。在天然PC 12细胞中仅发现痕量的NMDAR 1蛋白。然而，在用含有在巨细胞病毒启动子控制下的NMDAR 1C克隆的表达载体转染的PC 12细胞中检测到NMDAR 1蛋白的表达。这些结果表明，NMDAR 1蛋白在PC 12细胞中的表达可能是由转录后机制控制的。PC 12细胞系可作为研究NMDAR 1的转录、转录后和翻译调控的模型系统。此外，特定细胞类型中NMDAR 1 RNA的存在可能不一定表明NMDAR 1蛋白的表达。
Expression of RNA for the NMDAR1 subunit of the N-methyl-D-aspartate receptor was detected by Northern hybridization in both nerve growth factor-differentiated and undifferentiated rat pheochromocytoma (PC12) cells. The NMDA receptor type 1 (NMDAR1) message in PC12 cells was similar in size to that expressed in hippocampal neurons. PC12 cell cDNAs that were amplified by polymerase chain reaction with primers flanking the coding region of NMDAR1 corresponded to the NMDAR1 splice variant NMDA receptor type 1 isoform C (NMDAR1C). Using calcium imaging or patch-clamp recording, no functional NMDA-gated ion channels were found in PC12 cells. A monoclonal antibody against NMDAR1 was developed in order to investigate whether or not NMDAR1 protein was present in PC12 cells. Only trace amounts of NMDAR1 protein were found in native PC12 cells. However, expression of NMDAR1 protein was detected in PC12 cells that were transfected with an expression vector containing an NMDAR1C clone under control of a cytomegalovirus promoter. These findings suggest that the expression of NMDAR1 protein in PC12 cells may be controlled by post-transcriptional mechanisms. The PC12 cell line may serve as a model system for the study of the transcriptional, post-transcriptional, and translational regulation of NMDAR1. Furthermore, the presence of NMDAR1 RNA in a particular cell type may not necessarily indicate expression of NMDAR1 protein.