The Extracellular Domain of Notch2 Increases Its Cell-Surface Abundance and Ligand Responsiveness during Kidney Development

Abstract

SUMMARY Notch2, but not Notch1, plays indispensable roles in kidney organogenesis and Notch2 haploinsufficiency is associated with Alagille syndrome. We proposed that proximal nephron fates are regulated by a threshold that requires nearly all available free Notch intracellular domains (ICDs), but we could not identify the mechanism explaining why Notch2 (N2) is more important than Notch1 (N1). By generating mice that swap their ICDs, we establish that overall protein concentration, expression domain, or ICD amino acid composition does not account for the differential requirement for these receptors. Instead, we find that the N2 extracellular domain (ECD) increases Notch protein localized to the cell surface during kidney development and is cleaved more efficiently upon ligand binding. This context-specific asymmetry in NICD release efficiency is further enhanced by Fringe. Our results indicate that elevating N1 surface level could compensate for the loss of N2 signal in specific cell contexts.

The intracellular domains of Notch1 and 2 are functionally equivalent during development and carcinogenesis

“…We discovered that in the kidney Notch2 constituted the larger proportion of Notch receptors at the cell surface and was cleaved in response to ligands more efficiently, and that these differences were coded by the amino acid composition of the extracellular domain (ECD). N1ICD and N2ICD were perfectly equivalent: 100% of nephrons were rescued by the N1ICD when it was expressed from the Notch2 locus, whereas no nephron formed when N2ICD was solely produced from the Notch1 locus (in Pax3-Cre, N2 f/f , N1 12/12 animals, which lack endogenous Notch2 in the developing kidney; see figure 3 of Liu et al, 2013). This mirrors the absolute requirement for Notch2 in kidney development (Cheng et al, 2007).…”

“…1A,B); reciprocally, cleavage of the Notch2 extracellular and transmembrane domain of the N21 allele releases the Notch1 ICD (N1ICD; Fig. 1A,B) (Liu et al, 2013). Using these strains, we analyzed kidney development and found no support for the hypothesis that NICD composition was driving the differences between the two receptors.…”

“…Motivated by these observations and by the apparent asymmetric functions of Notch1 and Notch2 in renal development (Cheng et al, 2007), we created two new strains of mice harboring rearranged Notch1 and Notch2 loci (Liu et al, 2013): Notch12 (N12) and Notch21 (N21). In response to ligands, the Notch1 extracellular and transmembrane domain of the N12 allele is sequentially cleaved to release the Notch2 ICD (N2ICD; Fig.…”

“…Using these strains, we analyzed kidney development and found no support for the hypothesis that NICD composition was driving the differences between the two receptors. Instead, we identified a different mechanism that explained the divergent function of Notch1 and Notch2 in kidney development (Liu et al, 2013). We discovered that in the kidney Notch2 constituted the larger proportion of Notch receptors at the cell surface and was cleaved in response to ligands more efficiently, and that these differences were coded by the amino acid composition of the extracellular domain (ECD).…”

SpDamID: Marking DNA Bound by Protein Complexes Identifies Notch-Dimer Responsive Enhancers

“…Transient co-expression of SpDamID pairs from the bi-directional, Doxycycline (DOX)-responsive pBI Tet vector ( Figure S1C and S1D) will test for reconstituted DAM activity in mK4 cells (mouse kidney progenitor cell line, (Valerius et al, 2002)) stably expressing the Tet-On tetracycline responsive transactivator rtTA. These cells were chosen because Notch proteins have important functions in kidney progenitor cells via poorly defined targets (Cheng et al, 2007;Liu et al, 2013), and to allow us to analyze the impact of protein expression level on the signal-to-noise ratio. To further improve signal-to-noise ratio and to allow temporal control of SpDamID, we fused the AM domain with the estrogen receptor hormone-binding domain (Ert2).…”