Meier T, Perez G M, Wallace B G
Department of Physiology, University of Colorado Health Sciences Center, Denver 80262, USA.
J Cell Biol. 1995 Oct;131(2):441-51. doi: 10.1083/jcb.131.2.441.
Agrin induces the formation of highly localized specializations on myotubes at which nicotinic acetylcholine receptors (AChRs) and many other components of the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction accumulate. Agrin also induces AChR tyrosine phosphorylation. Treatments that inhibit tyrosine phosphorylation prevent AChR aggregation. To examine further the relationship between tyrosine phosphorylation and receptor aggregation, we have used the technique of fluorescence recovery after photobleaching to assess the lateral mobility of AChRs and other surface proteins in mouse C2 myotubes treated with agrin or with pervanadate, a protein tyrosine phosphatase inhibitor. Agrin induced the formation of patches in C2 myotubes that stained intensely with anti-phosphotyrosine antibodies and within which AChRs were relatively immobile. Pervanadate, on the other hand, increased protein tyrosine phosphorylation throughout the myotube and caused a reduction in the mobility of diffusely distributed AChRs, without affecting the mobility of other membrane proteins. Pervanadate, like agrin, caused an increase in AChR tyrosine phosphorylation and a decrease in the rate at which AChRs could be extracted from intact myotubes by mild detergent treatment, suggesting that immobilized receptors were phosphorylated and therefore less extractable. Indeed, phosphorylated receptors were extracted from agrin-treated myotubes more slowly than nonphosphorylated receptors. AChR aggregates at developing neuromuscular junctions in embryonic rat muscles also labeled with anti-phosphotyrosine antibodies, suggesting that tyrosine phosphorylation could mediate AChR aggregation in vivo as well. Thus, agrin appears to induce AChR aggregation by creating circumscribed domains of increased protein tyrosine phosphorylation within which receptors become phosphorylated and immobilized.
聚集蛋白可诱导肌管上形成高度局部化的特化结构，脊椎动物骨骼肌神经肌肉接头处的烟碱型乙酰胆碱受体（AChRs）和许多其他突触后装置成分在此处聚集。聚集蛋白还可诱导AChR酪氨酸磷酸化。抑制酪氨酸磷酸化的处理可阻止AChR聚集。为进一步研究酪氨酸磷酸化与受体聚集之间的关系，我们使用了光漂白后荧光恢复技术，以评估在经聚集蛋白或过钒酸盐（一种蛋白酪氨酸磷酸酶抑制剂）处理的小鼠C2肌管中AChRs和其他表面蛋白的侧向流动性。聚集蛋白诱导C2肌管中形成斑块，这些斑块用抗磷酸酪氨酸抗体染色强烈，且AChRs在其中相对不移动。另一方面，过钒酸盐增加了整个肌管中的蛋白酪氨酸磷酸化，并导致弥漫分布的AChRs的流动性降低，而不影响其他膜蛋白的流动性。过钒酸盐与聚集蛋白一样，导致AChR酪氨酸磷酸化增加，以及通过温和去污剂处理从完整肌管中提取AChRs的速率降低，这表明固定化的受体被磷酸化，因此更难提取。实际上，从经聚集蛋白处理的肌管中提取磷酸化受体的速度比未磷酸化受体慢。胚胎大鼠肌肉发育中的神经肌肉接头处的AChR聚集体也用抗磷酸酪氨酸抗体标记，这表明酪氨酸磷酸化在体内也可能介导AChR聚集。因此，聚集蛋白似乎通过创建蛋白酪氨酸磷酸化增加的限定区域来诱导AChR聚集，在这些区域内受体被磷酸化并固定化。
