Robbins M J, Ciruela F, Rhodes A, McIlhinney R A
Medical Research Council Anatomical Neuropharmacology Unit, Oxford, England, UK.
J Neurochem. 1999 Jun;72(6):2539-47. doi: 10.1046/j.1471-4159.1999.0722539.x.
The metabotropic glutamate receptor mGluR1alpha in membranes isolated both from rat brain and from cell lines transfected with cDNA coding for the receptor migrates as a disulphide-bonded dimer on sodium dodecyl sulphate-polyacrylamide gels. Dimerization of mGluR1alpha takes place in the endoplasmic reticulum because it is not prevented by exposing transfected human embryonic kidney (HEK) 293 cells to the drug brefeldin A, a drug that prevents egress of proteins from the endoplasmic reticulum. Dimerization was also not dependent on protein glycosylation as it was not prevented by treatment of the cells with tunicamycin. Using a mammalian expression vector containing the N-terminal domain of mGluR1alpha, truncated just before the first transmembrane domain (NT-mGluR1alpha), we show that the N-terminal domain is secreted as a soluble disulphide-bonded dimeric protein. In addition, the truncated N-terminal domain can form heterodimers with mGluR1alpha when both proteins are cotransfected into HEK 293 cells. However, mGluR1alpha and its splice variant mGluR1beta did not form heterodimers in doubly transfected HEK 293 cells. These results show that although the N-terminal domain of mGluR1alpha is sufficient for dimer formation, other domains in the molecule must regulate the process.
从大鼠脑以及转染了编码该受体的cDNA的细胞系中分离出的膜中的促代谢型谷氨酸受体mGluR1α，在十二烷基硫酸钠-聚丙烯酰胺凝胶上以二硫键连接的二聚体形式迁移。mGluR1α的二聚化发生在内质网中，因为将转染的人胚肾（HEK）293细胞暴露于布雷菲德菌素A（一种阻止蛋白质从内质网流出的药物）并不能阻止二聚化。二聚化也不依赖于蛋白质糖基化，因为用衣霉素处理细胞并不能阻止二聚化。使用一个包含mGluR1α的N端结构域（在第一个跨膜结构域之前被截短，即NT-mGluR1α）的哺乳动物表达载体，我们发现该N端结构域作为一种可溶性的二硫键连接的二聚体蛋白被分泌。此外，当这两种蛋白共转染到HEK 293细胞中时，截短的N端结构域可以与mGluR1α形成异源二聚体。然而，mGluR1α及其剪接变体mGluR1β在双重转染的HEK 293细胞中不形成异源二聚体。这些结果表明，虽然mGluR1α的N端结构域足以形成二聚体，但分子中的其他结构域必定对这一过程有调控作用。
