Mah Stephanie J, Cornell Elizabeth, Mitchell Nicholas A, Fleck Mark W
Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York 12208, USA.
J Neurosci. 2005 Mar 2;25(9):2215-25. doi: 10.1523/JNEUROSCI.4573-04.2005.
The glutamate receptor (GluR) agonist-binding site consists of amino acid residues in the extracellular S1 and S2 domains in the N-terminal and M3-M4 loop regions, respectively. In the present study, we sought to confirm that the conserved ligand-binding residues identified in the AMPA receptor S1S2 domains also participate in ligand binding of GluR6 kainate receptors. Amino acid substitutions were made in the GluR6 parent at R523, T690, and E738 to alter their potential interactions with ligand. Mutant receptors were expressed in human embryonic kidney 293 cells, confirmed by Western blot analysis, and tested by [3H]kainate binding and patch-clamp recording. Each of the binding site mutations was sufficient to reduce [3H]kainate binding to undetectable levels and eliminate functional responses to glutamate or kainate. As with our studies of other nonfunctional mutants (Fleck et al., 2003), immunocytochemical staining and cell-surface biotinylation studies showed that the mutant receptors were retained intracellularly and did not traffic to the cell surface. Endoglycosidase-H digests and colocalization with endoplasmic reticulum (ER) markers demonstrated that the mutant receptors are immaturely glycosylated and retained in the ER. Immunoprecipitation, native PAGE, and functional studies confirmed that the GluR6-binding site mutants are capable of multimeric assembly, indicating their retention in the ER does not result from a gross protein folding error. Together, these results confirm the role of R523, T690, and E738 directly in ligand binding to GluR6 and further support our previous report that nonfunctional GluRs are retained intracellularly by a functional checkpoint in ER quality control.
谷氨酸受体（GluR）的激动剂结合位点分别由N端细胞外S1和S2结构域以及M3-M4环区域中的氨基酸残基组成。在本研究中，我们试图确认在AMPA受体S1S2结构域中鉴定出的保守配体结合残基也参与GluR6红藻氨酸受体的配体结合。在GluR6亲本的R523、T690和E738处进行氨基酸替换，以改变它们与配体的潜在相互作用。突变受体在人胚肾293细胞中表达，通过蛋白质印迹分析进行确认，并通过[3H]红藻氨酸结合和膜片钳记录进行测试。每个结合位点突变都足以将[3H]红藻氨酸结合降低到无法检测的水平，并消除对谷氨酸或红藻氨酸的功能反应。与我们对其他无功能突变体的研究一样（Fleck等人，2003年），免疫细胞化学染色和细胞表面生物素化研究表明，突变受体保留在细胞内，未转运到细胞表面。内切糖苷酶-H消化以及与内质网（ER）标记物的共定位表明，突变受体糖基化不成熟，并保留在内质网中。免疫沉淀、非变性聚丙烯酰胺凝胶电泳和功能研究证实，GluR6结合位点突变体能够进行多聚体组装，表明它们保留在内质网中并非由严重的蛋白质折叠错误导致。总之，这些结果证实了R523、T690和E738直接在GluR6的配体结合中的作用，并进一步支持了我们之前的报告，即无功能的GluRs通过内质网质量控制中的功能检查点保留在细胞内。
