Abstract
The hedgehog (HH) family of ligands plays an important instructional role in metazoan development. HH proteins are initially produced as approximately 45-kDa full-length proteins, which undergo an intramolecular cleavage to generate an amino-terminal product that subsequently becomes cholesterol-modified (HH-Np). It is well accepted that this cholesterol-modified amino-terminal cleavage product is responsible for all HH-dependent signaling events. Contrary to this model we show here that full-length forms of HH proteins are able to traffic to the plasma membrane and participate directly in cell-cell signaling, both in vitro and in vivo. We were also able to rescue a Drosophila eye-specific hh loss of function phenotype by expressing a full-length form of hh that cannot be processed into HH-Np. These results suggest that in some physiological contexts full-length HH proteins may participate directly in HH signaling and that this novel activity of full-length HH may be evolutionarily conserved.
Highlights
The hedgehog (HH) family of ligands plays an important instructional role in metazoan development
Our results show that sonic hedgehog (SHH)-U is taken up by PTC-expressing cells when SHH-U-expressing cells ther confirm that full-length SHH can itself activate the SHH are physically associated with PTC-expressing cells ϳ75% of pathway, we immunoprecipitated SHH-U using two unrelated the time (Fig. 3A, fourth row from top, and 3B) but not by GFP
Our results suggest that full-length unprocessed HH proteins exhibit substantial levels of activity
Summary
SHH Activity Assays—The full-length cDNA of human SHH (a gift from Dr Cliff Tabin) was cloned into pcDNA3.1. The various immunoprecipitates were washed twice with PBS and incubated with confluent cells expressing SHHdependent luciferase reporter genes overnight to assay SHH activity as described above or analyzed by immunoblotting. Homozygous hhac embryos, and heads of wt third instar hh-Gal4ϾUAS-hh-M4 larvae enriched in imaginal discs were dissected in ice-cold PBS and homogenized in lysis buffer (150 mM NaCl, 50 mM Tris, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.5) supplemented with a protease inhibitor mixture for 30 min on ice. The proteins were separated by SDS-PAGE and analyzed by immunoblotting with rabbit “Calvados” polyclonal anti-HH antibody [14] or mouse anti␣-tubulin monoclonal antibody (Sigma). The images were collected on a Zeiss LSM510 confocal microscope
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