Roehl H, Bosenberg M, Blelloch R, Kimble J
Department of Genetics, University of Wisconsin, Madison 53706, USA.
EMBO J. 1996 Dec 16;15(24):7002-12.
In Caenorhabditis elegans, the GLP-1 receptor acts with a downstream transcriptional regulator, LAG-1, to mediate intercellular signaling. GLP-1 and LAG-1 are homologs of Drosophila Notch and Su(H) respectively. Here, we investigate the functions of two regions of the GLP-1 intracellular domain: the ANK repeat domain, which includes six cdc10/ankyrin repeats plus flanking amino acids, and the RAM domain, which spans approximately 60 amino acids just inside the transmembrane domain. First, we demonstrate that both ANK and RAM domains interact with the LAG-1 transcription factor. The interaction between the ANK domain and LAG-1 is only observed in nematodes by a co-localization assay and, therefore, may be either direct or indirect. By contrast, the interaction between the RAM domain and LAG-1 is likely to be direct, since it is observed by co-precipitation of the proteins in vitro as well as by yeast two-hybrid experiments. Second, we demonstrate that the RAM domain, when expressed in nematodes without a functional ANK repeat domain, does not mimic the unregulated receptor in directing cell fates or interfere with signaling by endogenous components. Finally, we show in yeast that the ANK repeats are strong transcriptional activators. Furthermore, missense mutations that eliminate receptor activity also abolish transcriptional activation by the GLP-1 ANK repeats in yeast. We speculate that one possible function for the ANK repeat domain is to act as a transcriptional co-activator with LAG-1.
在秀丽隐杆线虫中，GLP-1受体与下游转录调节因子LAG-1共同作用，介导细胞间信号传导。GLP-1和LAG-1分别是果蝇Notch和Su(H)的同源物。在此，我们研究了GLP-1细胞内结构域的两个区域的功能：ANK重复结构域，其包含六个cdc10/锚蛋白重复序列以及侧翼氨基酸；RAM结构域，其跨越跨膜结构域内约60个氨基酸。首先，我们证明ANK和RAM结构域均与LAG-1转录因子相互作用。ANK结构域与LAG-1之间的相互作用仅通过共定位分析在 nematodes 中观察到，因此可能是直接的或间接的。相比之下，RAM结构域与LAG-1之间的相互作用可能是直接的，因为在体外蛋白质的共沉淀以及酵母双杂交实验中均观察到了这种相互作用。其次，我们证明，当在没有功能性ANK重复结构域的线虫中表达时，RAM结构域不会在指导细胞命运方面模拟不受调控的受体，也不会干扰内源性成分的信号传导。最后，我们在酵母中表明，ANK重复序列是强大的转录激活因子。此外，消除受体活性的错义突变也消除了酵母中GLP-1 ANK重复序列的转录激活作用。我们推测ANK重复结构域的一个可能功能是作为与LAG-1的转录共激活因子。
