Mapping sites responsible for interactions of agrin with neurons
Mapping sites responsible for interactions of agrin with neurons
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DOI：
10.1046/j.1471-4159.2002.01102.x
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发表时间：
2002-10-01
影响因子：
4.7
通讯作者：
Sanes, JR
中科院分区：
文献类型：
--
作者：
Burgess, RW;Dickman, DK;Sanes, JR
多结构域蛋白聚糖聚集蛋白是骨骼肌神经肌肉接头突触后分化的重要组织者。聚集蛋白在大脑中也很丰富，但它在大脑中的作用尚不清楚。作为理解这些作用的一步，我们绘制了负责神经元与聚集蛋白相互作用的位点。首先，我们使用了一系列的重组聚集蛋白片段，以显示聚集蛋白上至少有四个位点与鸡睫状神经元相互作用。阻断抗体和肽的使用表明，神经元通过α V β 1整联蛋白粘附于三个G结构域中第二个的位点，并通过不同的β 1整联蛋白粘附于四个表皮生长因子（EGF）重复序列中最后一个的位点。第三，整合素独立的粘附网站是附近，但不同的网站，在肌肉中诱导突触后分化。然而，这些结构域不足以解释全长聚集蛋白的轴突生长抑制特性，其由分子的N-末端的一半介导。然后，我们使用第二组聚集蛋白突变体来证明和映射在SN-异构体的聚集蛋白的氨基末端的跨膜结构域。聚集蛋白的细胞外基质结合形式，称为LN，具有分泌和结合层粘连蛋白所需的氨基末端。由神经元选择性表达的SN形式具有变体氨基末端，其将聚集蛋白从分泌的基质相关蛋白转化为II型跨膜蛋白，提供了在中央而不是神经肌肉突触裂隙中呈递聚集蛋白的机制。SN-氨基末端可以介导异源蛋白的外化和膜锚定，但不足以将它们靶向突触。总之，这些研究确定了有助于神经元聚集蛋白亚细胞定位和信号传导的位点。
The multidomain proteoglycan agrin is a critical organizer of postsynaptic differentiation at the skeletal neuromuscular junction. Agrin is also abundant in the brain, but its roles there are unknown. As a step toward understanding these roles, we mapped sites responsible for interactions of neurons with agrin. First, we used a series of recombinant agrin fragments to show that at least four sites on agrin interact with chick ciliary neurons. Use of blocking antibodies and peptides indicated that neurons adhere to a site in the second of three G domains by means of alphaVbeta1 integrin, and to a site in the last of four epidermal growth factor (EGF) repeats via a distinct beta1 integrin. A third, integrin-independent adhesion site is near to but distinct from the site that induces postsynaptic differentiation in muscles. These domains are insufficient, however, to account for neurite outgrowth-inhibiting properties of full-length agrin, which are mediated by the N-terminal half of the molecule. We then used a second set of agrin mutants to demonstrate and map a transmembrane domain in the amino-terminus of the SN-isoform of agrin. The extracellular matrix-bound form of agrin, called LN, bears an amino-terminus required for secretion and binding to laminin. The SN form, which is selectively expressed by neurons, bears a variant amino terminus that converts agrin from a secreted, matrix-associated protein to a type-II transmembrane protein, providing a mechanism for presenting agrin in central, as opposed to neuromuscular, synaptic clefts. The SN-amino terminus can mediate externalization and membrane anchoring of heterologous proteins, but is insufficient to target them to the synapse. Together, these studies define sites that contribute to the subcellular localization of and signaling by neuronal agrin.