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The netrin-G1 ligand NGL-1 promotes the outgrowth of thalamocortical axons

Abstract

Netrin-G1 is a lipid-anchored protein that is structurally related to the netrin family of axon guidance molecules. Netrin-G1 does not bind any of the known netrin receptors and its function is not known. Here we identify human netrin-G1 ligand (NGL-1), a transmembrane protein containing leucine-rich repeat (LRR) and immunoglobulin (Ig) domains that specifically interacts with netrin-G1 through its LRR region. Whereas netrin-G1 is expressed highly in mouse thalamic axons, NGL-1 is most abundant in the striatum and the cerebral cortex—the intermediate and final targets, respectively, of thalamocortical axons (TCAs). Surface-bound NGL-1 stimulates, but soluble NGL-1 disrupts, the growth of embryonic thalamic axons, and in vitro data indicate that NGL-1 activity may be mediated at least partially by netrin-G1. Our findings provide evidence that netrin-G1 functions as an important component of the NGL-1 receptor to promote TCA outgrowth and that membrane-bound netrins can participate in receiving axonal signaling pathways.

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Figure 1: Specific and direct interaction of NGL-1 and netrin-G1 on the cell surface.
Figure 2: Characterization of the interaction of NGL-1 and netrin-G1.
Figure 3: Structure of NGL-1 and related molecules.
Figure 4: Distribution of NGL-1 and netrin-G1 transcripts and endogenous NGL-1-binding activity.
Figure 5: Biological activity of NGL-1.

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Acknowledgements

We thank members of Rosenthal laboratory and colleagues at Rinat Neuroscience Corporation for discussion; and the sequencing and oligonucleotide synthesis laboratories at Genentech for technical support during the early phase of the investigation.

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Correspondence to John C Lin or Arnon Rosenthal.

Supplementary information

Supplementary Fig. 1.

NGL is not secreted from transfected COS7 cells. The cDNA of NGL with HA-tagged in the extracellular domain (lanes 2, 4) and that of a myc-tagged chicken netrin-1 (lanes 6, 8) were transfected into COS7 cells along with mock transfection controls (lanes 1, 3, 5, 7). Sixteen mL of the total cell lysates (lanes 1,2, 5, 6) and of the 30-fold concentrated supernatants (lanes 3, 4, 7, 8) were assayed by Western blot using anti-HA (lanes 1-4) or anti-myc antibody (lanes 5-8). In the NGL-HA transfected supernatant, no HA-tagged protein was detectable that is consistent with the predicted extracellular domain of NGL (~58.9 kDa). By contrast, secreted myc-tagged netrin was readily detected in the supernatant (lane 8). (PDF 67 kb)

Supplementary Fig. 2.

SSequence alignment of human, mouse and chicken NGL family members. The GenBank accession numbers of the sequences are AK032567 (mouse NGL), AF196976 (human NAG14), AF300458 (mouse NAG14), The chicken NGL protein sequence is derived from the partial cDNA cloned by RT-PCR (see supplemental methods). The identical amino acid residues are shaded in black and the conserved residues in gray. (PDF 23 kb)

Supplementary Fig. 3.

The mRNA expression pattern of the mouse NGL (A) and NAG14 homologues (B) are identical in the E14.5 mouse forebrain. The probe of mouse NGL used corresponds to nucleotides (nt) 2479-2776 of the GenBank entry AK032467; whereas the probe of the mouse NAG14 homologue corresponds to nt 1309-1604 of the GenBank entry AF300458. Each of these probes is only 58~60% identical to the corresponding region of the other mouse gene with no continuous stretch of identical sequence longer than 8 nucleotides. Abbreviations: Ctx, cerebral cortex; DT, dorsal thalamus; VT, ventral thalamus; Str, striatum (PDF 35 kb)

Supplementary Methods (PDF 9 kb)

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Lin, J., Ho, WH., Gurney, A. et al. The netrin-G1 ligand NGL-1 promotes the outgrowth of thalamocortical axons. Nat Neurosci 6, 1270–1276 (2003). https://doi.org/10.1038/nn1148

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