Roles of the RAM and ANK domains in signaling by the C-elegans GLP-1 receptor

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 similar to 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-focalization 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.