Acetylcholine receptor-aggregating activity of agrin isoforms and mapping of the active site.
Acetylcholine receptor-aggregating activity of agrin isoforms and mapping of the active site.
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DOI：
10.1083/jcb.128.4.625
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发表时间：
1995-02
期刊：
影响因子：
--
通讯作者：
Ruegg MA
中科院分区：
文献类型：
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作者：
Gesemann M;Denzer AJ;Ruegg MA
AGRIN是一种基膜蛋白，可诱导乙酰胆碱受体(AChRs)和其他分子在发育中的神经肌肉连接处聚集。鸡集聚蛋白mRNA在A和B两个位点的选择性剪接产生了八种可能的异构体，其中五种在体内表达。运动神经元表达高水平的异构体，在A和B位点插入，肌肉细胞合成在B位点缺乏氨基酸的异构体。为了进一步了解集聚蛋白诱导AChR聚集的机制，我们测定了每种集聚蛋白异构体和截断突变体的EC50(诱导AChR聚集的有效浓度)。在鸡肌管上，A4B8的COOH末端的95-kD片段的EC50约为35 pm，agrinA4B19的EC50约为110 pm，agrinA4B11的EC50约为5 nM。当用agrinA4B0的NM观察到一些AchR簇时，没有检测到agrinA0B0的活性。重组全长鸡集聚蛋白和100-kD的射线集聚蛋白具有相似的EC50值。AgrinA4B8的45-kD，COOH-末端片段保持高活性(EC50约等于130 pm)，21-kD片段仍有活性，但需要更高的浓度(EC50约等于13 nM)。与45kD片段不同，21kD片段既不与肝素结合，也不抑制其诱导AChR聚集的能力。这些数据定量地表明，在运动神经元中表达的agrinA4B8和agrinA4B19活性最高，而在肌肉细胞合成的主要亚型agrinA0B0中没有检测到活性。此外，我们的结果表明，包含B8位点和最具COOH末端G-like结构域的片段就足以进行这种活性，而与肝素结合所需的集聚蛋白结构域和AChR聚集所需的集聚结构域是不同的。
Agrin is a basal lamina protein that induces aggregation of acetylcholine receptors (AChRs) and other molecules at the developing neuromuscular junction. Alternative splicing of chick agrin mRNA at two sites, A and B, gives rise to eight possible isoforms of which five are expressed in vivo. Motor neurons express high levels of isoforms with inserts at sites A and B, muscle cells synthesize isoforms that lack amino acids at the B-site. To obtain further insights into the mechanism of agrin-induced AChR aggregation, we have determined the EC50 (effective concentration to induce half-maximal AChR clustering) of each agrin isoform and of truncation mutants. On chick myotubes, EC50 of the COOH-terminal, 95-kD fragment of agrinA4B8 was approximately 35 pM, of agrinA4B19 approximately 110 pM and of agrinA4B11 approximately 5 nM. While some AChR clusters were observed with 64 nM of agrinA4B0, no activity was detected for agrinA0B0. Recombinant full-length chick agrin and a 100-kD fragment of ray agrin showed similar EC50 values. A 45-kD, COOH-terminal fragment of agrinA4B8 retained high activity (EC50 approximately equal to 130 pM) and a 21-kD fragment was still active, but required higher concentrations (EC50 approximately equal to 13 nM). Unlike the 45-kD fragment, the 21-kD fragment neither bound to heparin nor did heparin inhibit its capability to induce AChR aggregation. These data show quantitatively that agrinA4B8 and agrinA4B19, expressed in motor neurons, are most active, while no activity is detected in agrinA0B0, the dominant isoform synthesized by muscle cells. Furthermore, our results show that a fragment comprising site B8 and the most COOH- terminal G-like domain is sufficient for this activity, and that agrin domains required for binding to heparin and those for AChR aggregation are distinct from each other.