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Hedgehog Signaling, Which Requires Two Transmembrane Proteins, Relieves Repression of Target Genes

The Hedgehog signal is secreted from cells as a 45-kDa precursor protein. Cleavage of this secreted precursor produces a 20-kDa N-terminal fragment, which is associated with the plasma membrane and contains the inductive activity, and a 25-kDa C-terminal fragment. A series of elegant experiments demonstrated how the N-terminal Hedgehog fragment, which does not contain any hydrophobic sequences, acquires an affinity for the membrane. As depicted in Figure 15-30,

▲ FIGURE 15-30 Processing of Hedgehog (Hh) precursor protein. Removal of the N-terminal signal peptide from the initial translation product yields the 45-kDa Hh precursor consisting of residues 83-471 in the original protein. Nucleophilic attack by the thiol side chain of cysteine 258 (Cys-258) on the carbonyl carbon of glycine 257 (Gly-257) forms a thioester intermediate. The C-terminal domain then catalyzes the formation of an ester bond between the p-3 hydroxyl group of cholesterol and glycine 257 cleaving the precursor into two fragments. The N-terminal signaling fragment (tan) retains the cholesterol moiety and is modified by the addition of a palmitoyl group to the N-terminus. These two hydrophobic anchors tether the signaling fragment to the membrane. [Adapted from J. A. Porter et al., 1996, Science 274:255.]

Findings from genetic studies in Drosophila indicate that two membrane proteins, Smoothened (Smo) and Patched (Ptc), are required to receive and transduce a Hedgehog signal to the cell interior. Smoothened has 7 membrane-spanning a helices, similarly to G protein-coupled receptors (Chapter 13). Patched is predicted to contain 12 transmembrane a helices and is most similar to Niemann-Pick C1 protein (NPC1). These proteins may act as pumps or transporters. As discussed in Chapter 18, NPC1 protein is necessary for normal intracellular movement of sterols through vesicle-trafficking pathways. In humans, mutations in the NPC1 gene cause a rare, autosomal recessive disorder marked by defects in the lysosomal handling of cholesterol.

Drosophila embryos with loss-of-function mutations in the smoothened or hedgehog genes have very similar pheno-types. Moreover, both genes are required to activate transcription of the same target genes (e.g., wingless) during embryonic development. Loss-of-function mutations in patched produce a quite different phenotype, one similar to the effect of flooding the embryo with Hedgehog. Thus Patched appears to antagonize the actions of Hedgehog and vice versa. These findings and analyses of double mutants suggest that, in the absence of Hedgehog, Patched represses target genes by inhibiting a signaling pathway needed for gene activation. The additional observation that Smoothened is required for the transcription of target genes in mutants lacking patched function places Smoothened downstream in the pathway. The binding of Hedgehog evidently prevents

Patched from blocking Smoothened action, thus activating the transcription of target genes.

The results of recent studies have shown that, in the absence of Hedgehog, Patched is enriched in the plasma membrane, but Smoothened is in internal vesicle membranes. When cells receive a Hedgehog signal, both Patched and Hedgehog move from the cell surface into internal vesicles, whereas Smoothened moves from internal vesicles to the surface. The similarity of Patched to Niemann-Pick C1 protein, the covalent joining of cholesterol to Hedgehog, and the ability of cholesterol analogs such as cyclopamine to block reception of a Hedgehog signal all suggest a possible link between sterol metabolism and Hedgehog signaling. Indeed, one interesting idea is that developmental regulation by the Hedgehog system evolved from earlier cell components needed to control vesicle composition and movement.

Figure 15-31 depicts a current model of the Hedgehog pathway. Although the signal-transduction mechanisms are only partly understood, the pathway includes a cytoplasmic complex of proteins consisting of Fused (Fu), a serine-threonine kinase; Costal-2 (Cos-2), a microtubule-associated kinesin-like protein; and Cubitis interruptus (Ci), a transcription factor. In the absence of Hedgehog, when Patched inhibits Smoothened, these three proteins form a complex that binds to microtubules in the cytoplasm. Proteolytic cleavage of Ci in this complex generates a Ci fragment that translocates to the nucleus and represses target-gene expression. In the presence of Hedgehog, which relieves the

Exterior

Ptc Smo

BiiiiS fipfii

/WVVV/l VUtfi ksiB

Cytosol

GCCXHDpka

Proteasomal cleavage

Proteasomal cleavage

\ Microtubules

\ Microtubules

Nucleus

CCi75]

WWM Target genes

Target genes

M FIGURE 15-31 Operational model of the Hedgehog (Hh) signaling pathway. (a) In the absence of Hh, Patched (Ptc) protein inhibits Smoothened (Smo) protein by an unknown mechanism. In the absence of Smo signaling, a complex containing the Fused (Fu), Costal-2 (Cos2), and Cubitis interuptus (Ci) proteins binds to microtubules. Ci is cleaved in a process requiring the ubiquitin/proteasome-related F-box protein Slimb, generating the fragment Ci75, which functions as a transcriptional repressor. (b) In the presence of Hh, inhibition of Smo by Ptc is relieved. Signaling from Smo causes hyperphosphorylation of Fu and Cos2, and disassociation of the Fu/Cos2/Ci complex from microtubules. This leads to the stabilization of a full-length, alternately modified Ci, which functions as a transcriptional activator in conjunction with CREB binding protein (CBP). The exact membrane compartments in which Ptc and Smo respond to Hh and function are unknown; Hh signal causes Ptc to move from the surface to internal compartments while Smo does the opposite. [After K. Nybakken and N. Perrimon, 2002, Curr. Opin. Genet. Devel. 12:503.]

inhibition of Smoothened, the complex of Fu, Cos-2, and Ci is not associated with microtubules, cleavage of Ci is blocked, and an alternatively modified form of Ci is generated. After translocating to the nucleus, this Ci form binds to the transcriptional coactivator CREB-binding protein (CBP), promoting the expression of target genes. In addition to these components, protein kinase A participates in controlling Hedgehog-responsive target genes, which become inappropriately active when protein kinase A is inactivated. Phos-phorylation of Ci by protein kinase A appears to stimulate the proteolytic cleavage of Ci.

Hedgehog signaling, which is conserved throughout the animal kingdom, functions in the formation of many tissues and organs. Mutations in components of the Hedgehog signaling pathway have been implicated in birth defects such as cyclopia, a single eye resulting from union of the right and left brain primordia, and in multiple forms of human cancer. I

Wnt Signals Trigger Disassembly of an Intracellular Complex, Releasing a Transcription Factor

As noted previously, the Drosophila segment-polarity gene wingless encodes a protein that belongs to the Wnt family of secreted signals. Inactivation of wingless causes segment-polarity defects very similar to those caused by the loss of hedgehog function. This observation is logical because Hedgehog and Wingless form a positive feedback loop, with each protein maintaining production of the other (see Fig ure 15-29). The first vertebrate Wnt gene to be discovered was a mouse gene called Wnt-1 (formerly int-1). Activation of int-1 by insertion of a mouse mammary tumor virus (MMTV) provirus leads to mammary cancer. Hence Wnt-1 is a proto-oncogene, a normal cellular gene whose inappropriate expression promotes the onset of cancer (Chapter 23). The word Wnt is an amalgamation of wingless, the corresponding fly gene, with int for MMTV integration.

Genetic studies in Drosophila and C. elegans, studies of mouse proto-oncogenes and tumor-suppressor genes, and studies of cell junction components have all contributed to identifying many components of the Wnt signal-transduction pathway. Like Hedgehog proteins, Wnt proteins are modified by the addition of a hydrophobic palmitate group near their N termini, which may tether them to the plasma membrane of secreting cells and limit their range of action. Wnt proteins act through two cell-surface receptor proteins: Frizzled (Fz), which contains seven transmembrane a helices and directly binds Wnt; and Lrp, which appears to associate with Frizzled in a Wnt signal-dependent manner, at least in frog embryos. Mutations in the genes encoding Wingless, Frizzled, or Lrp (called Arrow in Drosophila) all have similar effects on the development of embryos. Frizzled protein and the Smoothened protein in Hedgehog signaling have sequence similarities, and both bear some resemblance to the G protein-coupled receptors discussed in Chapter 13. To date, however, evidence for G protein involvement downstream of Smoothened or Frizzled remains indirect and not compelling.

A current model of the Wnt pathway is shown in Figure 15-32. The central player in intracellular Wnt signal transduction is called p-catenin in vertebrates and Armadillo in

^ FIGURE 15-32 Operational model of the Wnt signaling pathway. (a) In the absence of Wnt, the kinase GSK3 constitutively phosphorylates p-catenin. Phosphorylated p-catenin is degraded and hence does not accumulate in cells. Axin is a scaffolding protein that forms a complex with GSK3, p-catenin, and APC, which facilitates phosphorylation of p-catenin by GSK3 by an estimated factor of >20,000. The TCF transcription factor in the nucleus acts as a repressor of target genes unless altered by Wnt signal transduction. (b) Binding of Wnt to its receptor Frizzled (Fz) recruits Dishevelled (Dsh) to the membrane. Activation of Dsh by Fz inhibits GSK3, permitting unphosphorylated p-catenin to accumulate in the cytosol. After translocation to the nucleus, p-catenin may act with TCF to activate target genes or, alternatively cause the export of TCF from the nucleus and perhaps its activation in cytosol. [After R. T Moon et al., 2002, Science 296:644; see also The Wnt Gene Homepage, www.stanford.edu/~rnusse/wntwindow. html.]

(a) -Wnt Exterior

Frizzled (Fz)

(a) -Wnt Exterior

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