All Mammalian Hedgehog Proteins Interact with Cell Adhesion Molecule, Down-regulated by Oncogenes (CDO) and Brother of CDO (BOC) in a Conserved Manner

Abstract

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.Hedgehog (Hh) 2 signaling proteins mediate key cell differentiation and tissue patterning events during animal development (1-3). Hh proteins generate tissue pattern, and fit the classical definition of a morphogen, by forming concentration gradients emanating from sites of secretion and eliciting different cell fate responses at different concentrations (3, 4). 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. Shh is essential for normal development of the nervous system, skeleton, and limbs (3). Ihh and Dhh are required for normal skeletal and testis development, respectively (5, 6). Hh proteins also play a role in maintenance and regeneration of adult tissues and stem cells (7,8), and abnormal Hh signaling has been implicated in several cancers (9). Drugs targeting the Hh pathway are currently in late stage clinical trials for the treatment of basal cell carcinoma (10) and early stage trials for treatment of breast, pancreatic, ovarian, brain, and gastric cancers (10, 11).Hh distribution and responsiveness are tightly regulated, and many factors influence the secretion, movement, and reception of Hh signals (12, 13). Hh proteins are synthesized as 45-kDa precursors that cleave themselves to generate an N-terminal 19-kDa fragment (HhN) and a C-terminal 25-kDa fragment (HhC). HhC mediates this reaction, which also results in the covalent attachment of cholesterol to the C terminus of HhN (14). HhN is palmitoylated at its N terminus in a separate reaction, and dually lipidated HhN is secreted as part of a multivalent lipoprotein particle (13,15). HhN mediates all known Hh signaling activities, and HhN lipidation and multivalency are required both for full potency and to regulate the distribution of HhN (12). Reception of the Hh signal involves Patched (Ptc) (16), a 12-pass integral membrane protein with homology to proton-driven bacterial transporters, and Smoothened (17), a 7-pass integral membrane protein with homology to G-protein coupled receptors. In the absence of Hh, Ptc inhibits the activity of Smoothened (17). Hh relieves this inhibition, resulting in the activation of Smoothened and downstream effectors that converge on Gli family transcription factors to alter expression levels of target genes (17).Despite high sequence similarity between Hh proteins, not all interactions between Hh proteins and cell-surface receptors are conserved between vertebrates and invertebrates. Vertebrate Hh proteins appear to interact directly with cognate Ptc proteins, but a direct interaction between the N-terminal signaling domai...

Indian hedgehog mutations causing brachydactyly type A1 impair Hedgehog signal transduction at multiple levels

“…We confirmed that the three critical BDA1 mutations (p.E95K, p.D100E, and p.E131K) were within a negatively charged calcium-binding groove, as shown previously when mapped to the murine SHH structure [28,36]. Furthermore, a recent paper about the complex structure between IhhN and CDOFn 3 has demonstrated that this calcium-binding site is important for the interactions with Ihh partners [30]. The main-chain superimposition between IhhN and its complex with CDOFn 3 gave an RMSD of 0.268 Å, which was mainly contributed by the calcium-binding region.…”

“…Clearly, p.E95K altered the surface charge within this groove, changing it from negative to positive, without altering the vicinal structure. This alteration could impair the interaction with partner molecules through this groove and would be consistent with impaired PTC1, CDOFn 3 , and HIP1 interactions, as previously demonstrated [27,30], as it would alter the electrostatic interactions that are required. The E95 residue is likely to have an important role in the interaction with partners, as a p.E95G mutation and a specific deletion of this amino acid also leads to a BDA1 phenotype [21,22].…”

“…The main chain superimposition between human IhhN and murine ShhN gave a root mean square deviation (RMSD) of 0.202 Å, indicating a significant similarity. Further- [29,30]. However, IhhN shows a relatively low binding affinity for calcium, and bound calcium was not observed in either the WT IhhN or the mutant forms in our study, without the addition of calcium during the purification and crystallization process.…”

“…This mutation affected chondrocyte differentiation in the growth plate, delaying endochondral bone formation, and impaired the recruitment of mesenchymal cells into the distal cartilage in the developing digit, causing brachydactyly [27]. The E95K mutation maps to a calcium-binding groove in the X-ray crystal structure of murine SHH, a region that interacts with the PTC receptor and other partner proteins, including HIP and CDO [26,30]. There are three BDA1 mutations that line the surface of this calcium-binding groove.…”

Dally and Notum regulate the switch between low and high level Hedgehog pathway signalling

“…A subfamily of HSPGs, the glypicans, which are anchored to the plasma membrane by a glycosyl-phosphatidyl-inositol (GPI), might contribute to the reception of morphogens such as Hedgehog (Hh) (Gallet, 2011). HSPGs are not alone in this role, as other novel proteins have been implicated, including members of the Ihog/Boi (CDON/BOC) families; Ihog and Boi are essential for Hh signalling in flies Camp et al, 2010;Zheng et al, 2010) and BOC and CDON are essential Shh co-receptors in mammals (McLellan et al, 2008;Kavran et al, 2010;Allen et al, 2011;Izzi et al, 2011). Interestingly, these proteins interact with the extracellular matrix component heparin, which is required for the dimerisation of Ihog and its high-affinity interaction with Hh (McLellan et al, 2006).…”