Godfrey E W, Roe J, Heathcote R D
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
J Neurobiol. 2000 Sep 15;44(4):436-45.
Agrin is an extracellular synaptic protein that organizes the postsynaptic apparatus, including acetylcholine receptors (AChRs), of the neuromuscular junction. The COOH-terminal portion of agrin has full AChR-aggregating activity in culture, and includes three globular domains, G1, G2, and G3. Portions of the agrin protein containing these domains bind to different cell surface proteins of muscle cells, including alpha-dystroglycan (G1-G2) and heparan sulfate proteoglycans (G2), whereas the G3 domain is sufficient to aggregate AChRs. We sought to determine whether the G1 and G2 domains of agrin potentiate agrin activity in vivo, as they do in culture. Fragments from the COOH-terminal of a neuronal agrin isoform (4,8) containing G3, both G2 and G3, or all three G domains were overexpressed in Xenopus embryos during neuromuscular synapse formation in myotomal muscles. RNA encoding these fragments of rat agrin was injected into one-cell embryos. All three fragments increased the ectopic aggregation of AChRs in noninnervated regions near the center of myotomes. Surprisingly, ectopic aggregation was more pronounced after overexpression of the smallest fragment, which lacks the heparin- and alpha-dystroglycan-binding domains. Synaptic AChR aggregation was decreased in embryos overexpressing the fragments, suggesting a competition between endogenous agrin secreted by nerve terminals and exogenous agrin fragments secreted by muscle cells. These results suggest that binding of the larger agrin fragments to alpha-dystroglycan and/or heparan sulfate proteoglycans may sequester the fragments and inhibit their activity in embryonic muscle. These intermolecular interactions may regulate agrin activity and differentiation of the neuromuscular junction in vivo.
聚集蛋白是一种细胞外突触蛋白，可组织神经肌肉接头的突触后装置，包括乙酰胆碱受体（AChRs）。聚集蛋白的COOH末端部分在培养中具有完整的AChR聚集活性，包括三个球状结构域G1、G2和G3。含有这些结构域的聚集蛋白部分与肌肉细胞的不同细胞表面蛋白结合，包括α - 肌营养不良聚糖（G1 - G2）和硫酸乙酰肝素蛋白聚糖（G2），而G3结构域足以聚集AChRs。我们试图确定聚集蛋白的G1和G2结构域在体内是否像在培养中一样增强聚集蛋白的活性。在体节肌神经肌肉突触形成过程中，将含有G3、G2和G3或所有三个G结构域的神经元聚集蛋白同工型（4,8）COOH末端的片段在非洲爪蟾胚胎中过表达。将编码大鼠聚集蛋白这些片段的RNA注射到单细胞胚胎中。所有三个片段都增加了体节中央附近非神经支配区域AChRs的异位聚集。令人惊讶的是，最小片段（缺乏肝素和α - 肌营养不良聚糖结合结构域）过表达后异位聚集更为明显。在过表达这些片段的胚胎中，突触AChR聚集减少，这表明神经末梢分泌的内源性聚集蛋白与肌肉细胞分泌的外源性聚集蛋白片段之间存在竞争。这些结果表明，较大的聚集蛋白片段与α - 肌营养不良聚糖和/或硫酸乙酰肝素蛋白聚糖的结合可能会隔离这些片段并抑制它们在胚胎肌肉中的活性。这些分子间相互作用可能在体内调节聚集蛋白的活性和神经肌肉接头的分化。
