Sonic hedgehog protein signals not as a hydrolytic enzyme but as an apparent ligand for Patched

Abstract

The amino-terminal signaling domain of the Sonic hedgehog secreted protein (Shh-N), which derives from the Shh precursor through an autoprocessing reaction mediated by the carboxyl-terminal domain, executes multiple functions in embryonic tissue patterning, including induction of ventral and suppression of dorsal cell types in the developing neural tube. An apparent catalytic site within Shh-N is suggested by structural homology to a bacterial carboxypeptidase. We demonstrate here that alteration of residues presumed to be critical for a hydrolytic activity does not cause a loss of inductive activity, thus ruling out catalysis by Shh-N as a requirement for signaling. We favor the alternative, that Shh-N functions primarily as a ligand for the putative receptor Patched (Ptc). This possibility is supported by new evidence for direct binding of Shh-N to Ptc and by a strong correlation between the affinity of Ptc-binding and the signaling potency of Shh-N protein variants carrying alterations of conserved residues in a particular region of the protein surface. These results together suggest that direct Shh-N binding to Ptc is a critical event in transduction of the Shh-N signal.

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

“…Stock cultures of the stable-expression PTC1 EcR-CHO cells were maintained in complete medium supplemented with 400 µg/ml G418. Assays of 32 P-IhhN binding to cells expressing Ptc-CTD140 with competition by IhhN variants were conducted as described by Fuse et al [7]. PTC1 expression was induced in cloned stable derivatives of the cell line EcR-CHO by the addition of ponasterone A.…”

“…The proteins were subjected to PTC1 binding or Hh-signaling induction (see below). 32 P-labeled IhhN protein was prepared as described previously [7], using the catalytic subunit of protein kinase A (Sigma), [γ-32 P] ATP, and recombinant IhhN protein containing a protein kinase A site (RRASV) at the C-terminus of IhhN. To generate stable cell lines, EcR-CHO cells (Invitrogen) were transfected with the pIND-Ptc-CTD140 construct, and several independent clones were isolated for growth in 10% FBS and 800 µg/ml G418.…”

“…Hedgehog signaling occurs via binding to the receptor Patched (PTC) on the receiving cells, and the signal is transduced through Smoothened (SMO) and the GLI complex, resulting in the activation of target genes, such as Ptc1, Gli1, and Hip1 [7,8]. The range of Hh signaling is thought to be facilitated by its interaction with heparan sulfate proteoglycans (HSPGs) in the extracellular matrix [9][10][11] and the formation of a soluble multimeric complex where the lipid moieties are buried to facilitate movement [12][13][14].…”

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

“…Vertebrate Hh proteins appear to interact directly with cognate Ptc proteins, but a direct interaction between the N-terminal signaling domain of Drosophila Hh (dHhN) and Drosophila Ptc (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).…”

“…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).…”