Mechanistic Insights into the Generation and Transduction of Hedgehog Signaling

Abstract

Cell differentiation and proliferation require Hedgehog (HH) signaling and aberrant HH signaling causes birth defects or cancers. In this signaling pathway, the N-terminally palmitoylated and C-terminally cholesterylated HH ligand is secreted into the extracellular space with help of the Dispatched-1 (DISP1) and Scube2 proteins. The Patched-1 (PTCH1) protein releases its inhibition of the oncoprotein Smoothened (SMO) after binding the HH ligand, triggering downstream signaling events. In this review, we discuss the recent structural and biochemical studies on four major components of the HH pathway: the HH ligand, DISP1, PTCH1, and SMO. This research provides mechanistic insights into how HH signaling is generated and transduced from the cell surface into the intercellular space and will aid in facilitating the treatment of HH-related diseases.

Keywords: Dispatched; Hedgehog; Patched; Smoothened; signal transduction; sterol.

Figure 1. Key proteins and important processes in vertebrate HH signaling pathway.

(A) The production and trafficking of HH protein. The precursor of HH is auto-cleaved and HH-N is covalently modified by a cholesterol at the C-terminus; then HH-N is further palmitoylated by HHAT on ER membrane. After these processes, DISP1, a membrane protein located on the cell surface, facilitates the release of HH-N into the extracellular space from the producing cells. Scube2, an extracellular protein assists in the trafficking of HH-N in the extracellular space. In the HH receiving cells, before binding HH-N, the receptor PTCH1 localizes to the cilia and suppress the downstream protein SMO, keeping the GLI transcription factors in an inactive form (GLI-R). (B) The HH signal transduction from PTCH1 to SMO. The HH binding to PTCH1 relieves the inhibition of PTCH1 to SMO, causing SMO to relocate to the cilia. The activated SMO can promote the maturation of GLI to its active form (GLI-A). GLI-A enters the nuclei to turn on the HH signal.

Figure 2. Secondary Structures of human DISP1 (hDISP1) and human Scube2 (hScube2).

DISP1 shares a similar structure with PTCH1, consisting of 12 TMs, two ECDs, and N-terminal and C-terminal cytosolic flexible regions. The ECDs are involved in HH-N binding (indicated by arrows). A proprotein convertase named Furin can cleave the ECD-I (cleavage site is labeled) of DISP1 triggering HH-N release. TMs 2–6 form an SSD and mutations in the SSD affect the release of HH-N. Scube2 protein works in the extracellular space and consists of 9 EGF-like repeats, three CRDs, and one CUB domain, which is essential for HH-N binding.

Figure 3. Structures of PTCH1–HH complexes

(A) Structures of HH with CDO (PDB ID: 3D1M), BOC (PDB ID: 3N1M) and 5E1 antibody (PDB ID: 3MXW). CDO and BOC are HH-N co-receptors that up-regulate HH signaling; 5E1 down-regulates HH signaling. They bind HH-N via the calcium-mediated interface. (B) Structure of PTCH1 with native HH-N (PDB ID: 6OEV). At 1:1 molar ratio, the palmitate moiety of native HH-N inserts in to the ECDs of PTCH1, forming the palmitate-dominated interface. (C) Structure of PTCH1 with His-tagged HH-N (PDB ID: 6DMY). His-tagged HH-N lacks the palmitate modification and binds PTCH1 through the calcium-mediated interface in presence of Ca²⁺. (D) Structure of 2:1 PTCH-1 and native HH-N complex (PDB ID: 6E1H). PTCH1 and native HH-N were incubated at 2:1 molar ratio with 1 mM Ca²⁺. In this structure, native HH-N employs both interfaces to bind two PTCH1 molecules. The sterol-like molecules that are observed in the cryo-EM map shown in red mesh. They are located in both TMs and ECDs. (E) The potential insertion of cholesterol modification of HH-N into PTCH1 (PDB ID: 6RVD). The cholesterol modification of native HH-N may insert into the ECD-I of PTCH1, but further structural and functional data are needed to prove this hypothesis. (F) SHH-N C-terminal loop and cholesterol. The cryo-EM maps of the linker region between HH-N and its cholesterol modification that were refined by FrealignX or RELION-3 at different signal levels. Although the overall EM maps are almost the same, densities of the linker region are quite different due to refinements by different software. This suggests the densities at this region may not be suitable for building the structural model. The palmitate is shown in magenta sticks. Cholesterol is shown in red sticks. The calcium and zinc ions were shown in gray balls.

Figure 4. The putative sterol transport tunnel in PTCH1

(A) and (B) The putative sterol tunnel in PTCH1. A tunnel across PTCH1 was predicted based on protein structure. PTCH1 may transport sterols through this tunnel. Nanobody Tl23 squeezes the tunnel and may inhibit the activity of PTCH1. The tunnel is indicated in gray and the two gates in the transmembrane domain are indicated. Tl23 (green) binding site is colored in magenta. (C) Model of PTCH1-mediated sterol transport. PTCH1 may transport the sterol from the inner leaflet to outer leaflet of the cell membrane.

Figure 5. Model of HH signaling generation

In the absence of HH protein, PTCH1 locates on the cilia membrane and transports sterols out of the membrane to keep a low sterol concentration in cilia. After the palmitate-dominated interface of HH-N binds to PTCH1, the bound PTCH1 is inhibited. Then, the calcium-mediated interface of the HH-N can bind to the other PTCH1 molecule. These interactions generate the 2:1 complex, triggering an endocytosis of this complex to create a PTCH1-free cilia environment for the future accumulation of free cholesterol and other sterols. Then, SMO relocates to the cilia membrane to obtain the sterols for activating the HH signaling. Cholesterol and sphingomyelin (SM) are shown in pink and green sticks. PTCH1, HH and SMO are colored in blue, yellow and orange, respectively.

Figure 6. Structures of SMO with distinct ligands.

(A) Multiple ligand-binding sites in SMO. As a member of class-F GPCR, SMO has multiple ligand binding sites in 7-TMs and one additional site in the CRD. Recent structural studies have revealed the binding details of numerous ligands including both agonists and antagonists. (B) Structure of xSMO with cyclopamine (PDB ID: 6D32). Compared with hSMO, the CRD of this xSMO structure undergoes a dramatic rotation, representing a possible active state of the CRD. (C) Structure of mSMO with SAG21k, cholesterol and a nanobody (Nb) (PDB ID: 6O3C). The mSMO is in an active conformation and the cholesterol molecule binds in the lower site of 7-TMs. The conformation of the CRD is similar with the inactive hSMO. (D) The Structure of hSMO with heterotrimeric G_i in the presence of 24(S),25-EC (PDB ID: 6OT0). The hSMO is in an active conformation and the 24(S),25-EC binding site is higher than that of cholesterol in mSMO structure. The CRD density in this cryo-EM structure is not observed. The right panel shows the position comparison of the ligands in panel (C) and (D). The ligands are shown in sticks with different colors as in C and D.