Stone D M, Nikolics K
Department of Neuroscience, Genentech, Inc., South San Francisco, California 94080, USA.
J Neurosci. 1995 Oct;15(10):6767-78. doi: 10.1523/JNEUROSCI.15-10-06767.1995.
Agrin is an extracellular matrix protein that mediates the nerve-induced clustering of nicotinic acetylcholine receptors on target muscle cells, and thus plays a key role in development of the neuromuscular synapse. Alternative exon usage within the rat agrin gene predicts numerous protein isoforms, which differ by the inclusion or exclusion of small inserts at three sites in the C-terminal half of the molecule; the insert status at two of these sites, termed Y and Z, profoundly influences the acetylcholine receptor clustering activity. We have examined the cellular expression patterns of agrin messenger RNA transcripts during rat embryogenesis by in situ hybridization with isoform-specific probes. Six 36-mer oligonucleotide probes were designed to distinguish between mRNA isoforms at either the Y site: the encoded protein contains either no insert (Y0) or a 4-amino acid insert (Y4), or the Z site: the encoded protein contains either no insert or one of 8 (Z8), 11 (Z11), or 19 (Z19) amino acids. Strikingly different expression patterns were observed for the individual Y- and Z-site encoding messages. While optional exon usage predicts the possibility of eight different agrin isoforms at the two splice sites, we detected only four isoforms in vivo: Y4Z0, Y0Z0, Y4Z8, and Y4Z19. The Y4Z0 transcript, which comprised the majority of the agrin expressed, was localized exclusively to nervous tissue and exhibited a distribution profile suggestive of a potential role in neurogenesis and/or neural differentiation. From embryonic day 13 to birth, Y4Z0 was found in mitotic ventricular zones, spinal, cranial, and sympathetic ganglia, and diffusely throughout the brain. In contrast, Y0Z0 was not expressed in neurons, but specifically labeled capillary endothelial cells within the developing nervous system. Y4Z8 and Y4Z19, the forms most active in acetylcholine receptor aggregation, were expressed at low levels only in spinal and brainstem motor neurons; Z19 expression declined from embryonic day 15 to adulthood, whereas Z8 expression increased slightly during this period. Transcripts encoding the Z11 insert could not be detected. These data suggest potential novel biological roles for agrin beyond that originally proposed in synapse formation.
集聚蛋白是一种细胞外基质蛋白，可介导神经诱导的烟碱型乙酰胆碱受体在靶肌肉细胞上的聚集，因此在神经肌肉突触的发育中起关键作用。大鼠集聚蛋白基因内的可变外显子使用预测了众多蛋白质异构体，这些异构体因分子C端一半中三个位点处小插入片段的包含或排除而有所不同；其中两个位点（称为Y和Z）的插入状态深刻影响乙酰胆碱受体的聚集活性。我们通过用异构体特异性探针进行原位杂交，研究了大鼠胚胎发育过程中集聚蛋白信使核糖核酸转录本的细胞表达模式。设计了六种36聚体寡核苷酸探针，以区分Y位点（编码的蛋白质要么无插入片段（Y0），要么有一个4氨基酸插入片段（Y4））或Z位点（编码的蛋白质要么无插入片段，要么有8（Z8）、11（Z11）或19（Z19）个氨基酸中的一种）处的信使核糖核酸异构体。观察到单个Y和Z位点编码信息的表达模式有显著差异。虽然可变外显子使用预测了在两个剪接位点有八种不同集聚蛋白异构体的可能性，但我们在体内仅检测到四种异构体：Y4Z0、Y0Z0、Y4Z8和Y4Z19。构成大部分表达的集聚蛋白的Y4Z0转录本仅定位于神经组织，并呈现出一种分布模式，提示其在神经发生和/或神经分化中可能发挥作用。从胚胎第13天到出生，Y4Z0存在于有丝分裂的脑室区、脊髓、颅神经节和交感神经节中，并广泛分布于整个大脑。相比之下，Y0Z0在神经元中不表达，但特异性标记发育中的神经系统内的毛细血管内皮细胞。在乙酰胆碱受体聚集方面最活跃的Y4Z8和Y4Z19形式仅在脊髓和脑干运动神经元中低水平表达；Z19的表达从胚胎第15天到成年期下降，而Z8的表达在此期间略有增加。无法检测到编码Z11插入片段的转录本。这些数据表明集聚蛋白除了最初提出的在突触形成中的作用外，可能还有新的生物学作用。
