Regulation of Hedgehog Signalling Inside and Outside the Cell

Abstract

The hedgehog (Hh) signalling pathway is conserved throughout metazoans and plays an important regulatory role in both embryonic development and adult homeostasis. Many levels of regulation exist that control the release, reception, and interpretation of the hedgehog signal. The fatty nature of the Shh ligand means that it tends to associate tightly with the cell membrane, and yet it is known to act as a morphogen that diffuses to elicit pattern formation. Heparan sulfate proteoglycans (HSPGs) play a major role in the regulation of Hh distribution outside the cell. Inside the cell, the primary cilium provides an important hub for processing the Hh signal in vertebrates. This review will summarise the current understanding of how the Hh pathway is regulated from ligand production, release, and diffusion, through to signal reception and intracellular transduction.

Keywords: Hedgehog; heparan sulfate; regulation; signalling.

Figure 1

The sonic hedgehog (Shh) ligand is initially synthesised as a precursor with two distinct domains: the N-terminal “hedge” and C-terminal “hog” domain, which undergoes autoproteolysis to give N- and C-terminal fragments. The C-terminal fragment acts as a cholesterol transferase to attach cholesterol to the N-terminal fragment. Skinny hedgehog attaches palmitate to the N-terminus of hedgehog (Hh) to give rise to the fully processed form. Following synthesis, Hh is delivered from the endoplasmic reticulum to the cell membrane, where it is released from the cell via a number of different mechanisms. (1) Hh binds Disp in a cholesterol-dependent manner, and through the combined action of Disp and Scube2, is released from the cell. Heparan sulfate proteoglycans (HSPGs) act as assembly points for multiple components. Hh monomers are able to form multimeric complexes aided by association with HSPGs and Scube2. Hh is released following proteolytic processing by sheddases, which remove cholesterol and palmitate. These complexes are more soluble than the monomeric form and so are able to diffuse away from the cell; (2) Unprocessed Hh may re-enter the cell in a Disp-dependent fashion (2a) and be internalised by endosomal sorting complexes required for transport (ESCRT) proteins, which sort Hh proteins into intra-luminal vesicles (2b); These vesicles subsequently fuse with the plasma membrane and are released from the cell (2c); (3) Association of Hh with HSPGs results in loading of Hh into lipoprotein particles. Glypicans (Glycosylphosphatidylinositol (GPI)-linked HSPGs) may be cleaved and released along with Hh.

Figure 2

(Left) In the absence of hedgehog ligand, patched (PTC)—which is enriched in the cilial membrane—acts to repress smoothened (SMO) through the recruitment of lipoproteins and regulation of their phospholipid composition. SMO is maintained in its inactive state and sequestered within the cell. Upon exiting the cilium, GLI2 and GLI3 are phosphorylated by glycogen synthase kinsase 3β (GSK3β), casein kinase I (CKI), and protein kinase A (PKA); the activity of PKA is promoted by elevated cAMP levels due to the presence of G-protein-coupled receptor 161 GPR161. Phosphorylated GLI is recognised by β-TRCP, which promotes ubiquitylation and degradation of its C-terminal domain, giving rise to a cleaved repressor form. This cleaved repressor translocates to the nucleus and represses hedgehog target genes. GLI1 is not cleaved but is sequestered within the cytoplasm by suppressor of fused (SUFU), preventing it from activating downstream signalling; (Right) Upon binding of hedgehog, the hedgehog-PTC complex is internalised, and SMO inhibition by PTC is released; endocannabinoid levels are reduced, while phosphatidylinositol-4 phosphate (PI(4)P) levels increase, promoting SMO accumulation at the membrane. SMO is phosphorylated by CK1 and GRK2, leading to its activation. Activated SMO accumulates within the cilial membrane and binds EVC and EVC2. GPR161 exits the cilium and is internalised. GLI proteins within the cilial tip dissociate from SUFU and translocate the nucleus to activate Shh target genes.