Structure of a heparin-dependent complex of Hedgehog and Ihog

Abstract

H edgehog (Hh) is a secreted signaling molecule that mediates key tissue-patterning events during both vertebrate and invertebrate development (1-3). Hh also plays an important role in the maintenance and regulation of stem cells in adult organisms (4, 5). Abnormal activation of the Hh signaling pathway has been implicated in the initiation and growth of many human tumors (6), and drugs targeting the Hh pathway are under development (7).Hh is secreted but undergoes two lipid modifications that restrict its free diffusion and facilitate transport to appropriate target sites (8,9). Hh binding to Ptc, a 12-pass integral membrane protein with homology to bacterial resistance-nodulationdivision (RND) transporters (10) is a central event in Hh signaling and blocks the ability of Ptc to inhibit the seven-pass integral membrane protein Smoothened (Smo), a positive regulator of Hh responses (10). Recent genetic and RNAi experiments implicate the membrane-associated proteins Ihog and dally-like protein (Dlp) in Hh responsiveness (11)(12)(13)(14). Dlp, a member of the glypican family of heparan sulfate proteoglycans (HSPGs) (15), has dual roles in mediating Hh responsiveness and in transport of the Hh signal to distant cells (12)(13)(14). Heparan sulfate glycosaminoglycan chains, in particular, have also been implicated in Hh movement by the observation that tout velu, which encodes a heparan sulfate copolymerase (16, 17) that acts on Dlp (12), is required for normal Hh transport.Ihog, a Drosophila protein previously known as CG9211, is a type I transmembrane protein with four Ig domains followed by two FNIII domains, a membrane-spanning region, and a cytoplasmic region of no known function (11). Ihog is homologous to another Drosophila protein, CG32796 or brother of Ihog (BOI), and two mammalian proteins, CDO and BOC (18,19), that are also components of the Hh signaling pathway (11,20,21). Several observations indicate that Ihog may function as a coreceptor for Hh: (i) reduction of Ihog expression results in diminished Hh binding and responsiveness, (ii) epistasis experiments place Ihog function upstream or at the level of Ptc, (iii) the Ihog extracellular region is able to pull down HhN from conditioned medium, and (iv) coexpression of Ptc and Ihog results in a synergistic increase in Hh binding to the cell surface (11).The first FNIII domain of Ihog (IhogFn1) is necessary and sufficient to pull down HhN, but both FNIII domains are needed to synergize with Ptc and reconstitute Ihog function in cell-based signaling assays (11). Complicating interpretation of Ihog function is the observation that purified HhN and Ihog extracellular regions do not appreciably interact (see Fig. 2 A), indicating that a binary interaction between Ihog and Hh cannot explain Ihog function. To investigate the role of Ihog in Hh signaling, we initiated structural and biophysical studies of functional fragments of Drosophila melanogaster Ihog and Hh. Results and DiscussionStructure of Ihog FNIII Domains. The crystal structure of IhogFn1 was determ...

Mutational Analysis of Norrin-Frizzled4 Recognition

“…For basic fibroblast growth factor, heparin plays a critical role in receptor binding by functioning as a co-ligand (47, 54 -57). A role for heparin as a co-ligand has recently been demonstrated biochemically and crystallographically in a second signaling system; high affinity binding of Hedgehog to its transmembrane binding partner Ihog requires the formation of a ternary complex with heparin (58). The observation that heparin strongly enhances Norrin-CRD binding indicates that Norrin-extracellular matrix interactions not only control the spatial localization of Norrin but also play an intimate part in the Norrin-Fz4 signaling complex.…”

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

“…The specific crystal lattice contact in the original ShhN structure that was postulated as a possible Hh oligomerization interface involves Arg 73 (Lys 132 in Drosophila HhN) (23). This interface buries only 180 Å 2 of surface area and does not occur in any other HhN-containing crystal structures, either alone or complexed with Ihog, CDOFn3, BOCFn3, or Hip (22,32,36,37) and thus seems unlikely to represent a conserved self-interaction among HhN molecules.…”

“…The set of HhN structures analyzed includes both vertebrate and invertebrate HhNs; HhN in the presence or absence of calcium; and HhN in the presence or absence of the binding partners Ihog, CDO, BOC, and Hip (22,32,36,37) (supplemental Table 2). HhN-HhN contacts in each of 14 unique crystal lattices were analyzed, which yielded roughly 100 pairwise HhN interactions for subsequent analysis.…”

Hedgehogs like it sweet, too

“…Upon heparin binding, AT undergoes a conformational change, which increases AT's affinity for heparin. An allosteric mechanism can be ruled out for the Hh-Ihog signaling system, however, because McLellan et al (1) did not notice any conformational change in HhN or Ihog subsequent to binding.…”

“…Deregulated Hh signaling has been implicated in basal-cell carcinoma and medulloblastoma cancers (reviewed in ref. Although the cocrystallized heparin (a highly sulfated HSGAG) molecules are disordered in the structure, Leahy and coworkers (1) infer that HS plays a direct role in the assembly of the Hh signaling apparatus. Regions of positive electrostatic potential on Hh and Ihog are juxtaposed in the structure and form a continuous basic strip where HSGAG is proposed to bind and enhance Hh-Ihog affinity (Fig.…”