The mode of Hedgehog binding to Ihog homologues is not conserved across different phyla
✍ Scribed by McLellan, Jason S.; Zheng, Xiaoyan; Hauk, Glenn; Ghirlando, Rodolfo; Beachy, Philip A.; Leahy, Daniel J.
- Book ID
- 109897749
- Publisher
- Nature Publishing Group
- Year
- 2008
- Tongue
- English
- Weight
- 725 KB
- Volume
- 455
- Category
- Article
- ISSN
- 0028-0836
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✦ Synopsis
Hedgehog (Hh) proteins specify tissue pattern in metazoan embryos by forming gradients that emanate from discrete sites of expression and elicit concentration-dependent cellular differentiation or proliferation responses 1,2 . Cellular responses to Hh and the movement of Hh through tissues are both precisely regulated, and abnormal Hh signalling has been implicated in human birth defects and cancer . Hh signalling is mediated by its amino-terminal domain (HhN), which is dually lipidated and secreted as part of a multivalent lipoprotein particle . Reception of the HhN signal is modulated by several cell-surface proteins on responding cells, including Patched (Ptc), Smoothened (Smo), Ihog (known as CDO or CDON in mammals) and the vertebrate-specific proteins Hip (also known as Hhip) and Gas1 (ref. 11). Drosophila Ihog and its vertebrate homologues CDO and BOC contain multiple immunoglobulin and fibronectin type III (FNIII) repeats, and the first FNIII repeat of Ihog binds Drosophila HhN in a heparindependent manner . Surprisingly, pull-down experiments suggest that a mammalian Sonic hedgehog N-terminal domain (ShhN) binds a non-orthologous FNIII repeat of CDO . Here we report biochemical, biophysical and X-ray structural studies of a complex between ShhN and the third FNIII repeat of CDO. We show that the ShhN-CDO interaction is completely unlike the HhN-Ihog interaction and requires calcium, which binds at a previously undetected site on ShhN. This site is conserved in nearly all Hh proteins and is a hotspot for mediating interactions between ShhN and CDO, Ptc, Hip and Gas1. Mutations in vertebrate Hh proteins causing holoprosencephaly and brachydactyly type A1 map to this calcium-binding site and disrupt interactions with these partners.CDO is composed of five immunoglobulin domains followed by three FNIII repeats, a membrane-spanning region and a cytoplasmic tail . The second FNIII repeat of CDO (CDOFn2) is homologous to the Ihog repeat that binds Drosophila HhN and heparin, but the third FNIII repeat (CDOFn3) seems to be most important for binding Shh 12,14 (Figs ). However, purified CDOFn2, CDOFn3 and a tandem repeat of these domains (CDOFn23) all failed to interact appreciably with ShhN in the presence or absence of heparin, as judged by size-exclusion chromatography (SEC). Pull-down experiments showing an interaction between ShhN and CDOFn3 were performed in the presence of serum , and a search for additional factors involved in ShhN-CDO binding led to the discovery that calcium ions promote high-affinity interactions between ShhN and CDOFn3 (Supplementary Fig. ). Analytical ultracentrifugation and isothermal titration calorimetry (ITC) studies demonstrate that the interaction between ShhN and CDOFn3 is 1:1, requires calcium, is not heparin-dependent, and has a dissociation constant of ,1.3 mM (Figs , and Supplementary Fig. ).