Notch subunit heterodimerization and prevention of ligand-independent proteolytic activation depend, respectively, on a novel domain and the LNR repeats

Abstract

Notch proteins are transmembrane receptors that participate in a highly conserved signaling pathway that regulates morphogenesis in metazoans. Newly synthesized Notch receptors are proteolytically cleaved during transit to the cell surface, creating heterodimeric mature receptors comprising noncovalently associated extracellular (N(EC)) and transmembrane (N) subunits. Ligand binding activates Notch by inducing two successive proteolytic cleavages, catalyzed by metalloproteases and gamma-secretase, respectively, that permit the intracellular portion of N to translocate to the nucleus and activate transcription of target genes. Prior work has shown that the presence of N(EC) prevents ligand-independent activation of N, but the mechanisms involved are poorly understood. Here, we define the roles of two regions at the C-terminal end of N(EC) that participate in maintaining the integrity of resting Notch receptors through distinct mechanisms. The first region, a hydrophobic, previously uncharacterized portion of N(EC), is sufficient to form stable complexes with the extracellular portion of N. The second region, consisting of the three Lin12/Notch repeats, is not needed for heterodimerization but acts to protect N from ligand-independent cleavage by metalloproteases. Together, these two contiguous regions of N(EC) impose crucial restraints that prevent premature Notch receptor activation.

FIG. 1.

(A) Schematic representation of hN1. N^EC, Notch extracellular subunit; N^TM, Notch transmembrane subunit; NRR, negative regulatory region; LNR, domain comprising the three LIN12/Notch repeats; N^ECC, C-terminal segment of N^EC; N^TMX, N^TM extracellular region; TM, transmembrane segment; ICN, intracellular Notch; TAD, transactivation domain; S1, furin cleavage site. (B) Truncated hN1 polypeptides used in these studies.

FIG. 2.

Recovery of hN1 heterodimeric complexes secreted into conditioned media. Notch1-derived polypeptides with FLAG and HA epitope tags were resolved by SDS-PAGE under reducing conditions and detected on Western blots. Antibodies used to detect NRR and N^TMX were anti-FLAG and anti-HA, respectively. (A) The NRR-N^TMX single-chain precursor (36 kDa), detected in whole-cell extracts by Western blot with anti-FLAG. (B) Noncovalently associated NRR (27 kDa) and N^TMX (8.4 kDa) subunits, coimmunoprecipitated (IP) from conditioned media with anti-FLAG or anti-HA as indicated, and detected by Western blot using anti-FLAG (top panel), or anti-HA (lower panel). (C) Dissociation of the heterodimer upon treatment with SDS. Coimmunoprecipitated NRR and N^TMX subunits were treated with 1% NP-40, or 0.025% SDS prior to Western blotting with anti-FLAG (top panel) or anti-HA (lower panel).

FIG. 3.

The N^ECC region is sufficient for formation of stable heterodimers with N^TMX. (A, B) N^ECC-N^TMX heterodimers were recovered from conditioned media by immunoprecipitation (IP) with either anti-FLAG or anti-HA and were subjected to SDS-PAGE under reducing conditions. Each subunit was then detected by Western blotting for its respective epitope tag. (Top panel) Detection of N^ECC with anti-FLAG; (bottom panel) detection of N^TMX with anti-HA. (A) hN1 heterodimers. (B) hN2 and mN3 heterodimers. The asterisk denotes a polypeptide of slightly smaller molecular weight than hN2 N^ECC that is not competent for association with N^TMX. (C) Sequence alignment of the N^ECC region of various Notch proteins up to the putative furin site (S1). Identical and conserved residues among the sequences are enclosed in black and gray boxes, respectively. H, human; x, Xenopus; m, mouse; r, rat; and zf, zebra fish.

FIG. 4.

(A) Purification of the N^ECC-N^TMX-H₆ precursor from hN2 after expression in bacteria. M, molecular mass standards; E, the N^ECC-N^TMX-H₆ uncleaved precursor after elution from nickel-NTA beads. (B) Denaturing HPLC chromatograms of the hN2 precursor (top) and N^ECC-N^TMX heterodimer (bottom). Samples of both the precursor and intact heterodimer (after cleavage with recombinant furin) were recovered in a single peak after gel filtration. An aliquot of the peak from each sample was further analyzed by denaturing reversed-phase HPLC chromatography using a C₁₈ column. The peaks corresponding to the precursor and each of the subunits are indicated.

FIG. 5.

Signaling activity of native hN1 and a series of hN1 N^EC deletion variants in CSL reporter gene assays. Luciferase assays were performed on U2OS cell lysates prepared from cells transfected in triplicate with 50 ng of empty pcDNA3 plasmid or plasmids encoding the indicated forms of Notch1, along with a luciferase reporter plasmid containing iterated CSL-binding site and an internal control plasmid expressing Renilla luciferase from a thymidine kinase promoter. Firefly luciferase activity, normalized for variation in Renilla luciferase activity, is expressed relative to the activity in extracts prepared from cells transfected with empty pcDNA3, which is arbitrarily set to a value of 1.

FIG. 6.

The LNR domain of hN1 confers resistance to proteolysis at the S2 site. (A) Schematic representation of the hN1 polypeptides used in the proteolysis experiments, noting the position of the myc epitope tag, the site of S2 cleavage, and the difference in polypeptide length between native N^TM and the product after S2 cleavage, denoted N^TM*. (B) The S2 proteolysis product N^TM* is generated when the LNR domain is deleted from hN1. U2OS cells transfected with the indicated hN1 variants were incubated in the presence of a presenilin inhibitor (DFP-AA). N^TM and N^TM* polypeptides were then recovered by immunoprecipitation with an anti-myc antibody, resolved by SDS-PAGE under reducing conditions, and detected by Western blotting using anti-myc.

FIG. 7.

A form of Notch1 lacking both ligand-binding EGF repeats and the LNR domain, N^EC-N^TM, causes abnormal T-cell development. Peripheral blood was obtained from mice (n = 9 for each group) on day 54 after reconstitution with hematopoetic stem cells transduced with MigRI retroviruses encoding either NRR-N^TM, which lacks only the ligand-binding domain, or N^EC-N^TM. Representative flow cytometric results are shown.