Abstract
Hedgehog (Hh) signaling proteins stimulate cell proliferation, differentiation, and tissue patterning at multiple points in animal development. A single Hh homolog is present in Drosophila, but three Hh homologs, Sonic Hh, Indian Hh, and Desert Hh, are present in mammals. Distribution, movement, and reception of Hh signals are tightly regulated, and abnormal Hh signaling is associated with developmental defects and cancer. In addition to the integral membrane proteins Patched and Smoothened, members of the Drosophila Ihog family of adhesion-like molecules have recently been shown to bind Hh proteins with micromolar affinity and positively regulate Hh signaling. Cell adhesion molecule-related, down-regulated by oncogenes (CDO) and Brother of CDO (BOC) are the closest mammalian relatives of Drosophila Ihog, and CDO binds Sonic Hh with micromolar affinity and positively regulates Hh signaling. Despite these similarities, structural and biochemical studies have shown that Ihog and CDO utilize nonorthologous domains and completely different binding modes to interact with cognate Hh proteins. We report here biochemical and x-ray structural studies of Sonic, Indian, and Desert Hh proteins both alone and complexed with active domains of CDO and BOC. These results show that all mammalian Hh proteins bind CDO and BOC in the same manner. We also show that interactions between Hh proteins and CDO are weakened at low pH. Formation of Hh-mediated Hh oligomers is thought to be an important feature of normal Hh signaling, but no conserved self-interaction between Hh proteins is apparent from inspection of 14 independent Hh-containing crystal lattices.
Highlights
Grant R01HD055545. □S The on-line version of this article contains supplemental Tables 1–3 and additional references
Three Hh homologs are present in mammals: Sonic Hh (Shh), Indian Hh (Ihh), and Desert Hh (Dhh), but only a single Hh protein is present in Drosophila
We find that interactions between mammalian Hh proteins and CDO and Brother of CDO (BOC) are conserved in nature, and systematic inspection of 14 different HhN-containing crystal lattices reveals no evidence for a conserved contact that may reflect a physiological self-interaction between Hh proteins
Summary
(dPtc) has not been found [16, 18]. Instead, the homologous adhesion-like molecules Ihog or Brother of Ihog appear to be essential components of the Drosophila Hh receptor [19]. Biochemical and crystallographic studies show that interactions between the N-terminal signaling domain of Shh (ShhN) and CDO are calcium-dependent and involve the membrane-proximal type III fibronectin (FNIII) repeat of CDO (CDOFn3). Interactions between dHhN and Ihog are heparin-dependent and involve the Ihog FNIII repeat N-terminal to the ortholog of the membraneproximal FNIII repeat (IhogFn1) [22] (Fig. 1) In both cases, interactions between HhN and the cognate Ihog or CDO domains occur with low micromolar affinity [22], sufficient to tether multivalent HhN particles tightly to the cell surface. To investigate the nature of interactions between mammalian Hh and CDO homologs as well as search for evidence of conserved self-interactions among Hh proteins, we undertook biochemical and crystallographic studies of Ihh and Dhh both alone and complexed with CDO or BOC. We show that interactions between ShhN and CDOFn3 are diminished at low pH, suggesting that CDO and BOC will release bound Hh proteins in low pH environments
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