The Role of Patched and Smoothened in Hedgehog Signaling

by Jeff Wakeford, Pauline Li, Lisa Ricketts
and Jennifer Brandon

During development, cells need to differentiate and perform different functions in order to permit the eventual formation of a mature organism. Receptors on cell surfaces play a leading role in mediating the ability of cells to respond to activating signals during the development of an organism (Nusse, 1996). Hedgehog (Hh) represents a family of signaling molecules that control decisions regarding development. In Drosophila, Hh is required in the segmentation of embryos as well as in the patterning of imaginal-disc outgrowth. In vertebrates, the protein Sonic Hedgehog (Shh), is essential for developmental patterning events (Chen and Struhl, 1996). The signal in vertebrates is responsible for polarization of the anterior-posterior axis of the developing limb-bud and is the signal originating from the notochord involved in determining ventral cell fate of the somites and neural tube. Furthermore, in Drosophila, Hh is required for wingless (wg) and decapentaplegic (dpp) expression, which are both located downstream of the hh gene (Nusse, 1996). The wingless pathway, described in Drosophila, is involved in specifying the dorso-ventral axis, whereas dpp is involved in anterior-posterior patterning. When expressed in the ectoderm, wg and dpp act as extracellular signals that determine cells to become imaginal-disc cells. The receptor for Hh has been proposed to be Patched (Ptc), whereas previously it was suspected that Smoothened (Smo) was the primary candidate for the Hh receptor (Stone et al., 1996). The receptor may be covalently modified by Hh, indicating that the Hh protein may possess enzymatic activities such as proteolytic capabilities. Moreover, the Hh protein is involved in short-range posterior-anterior signaling that induces anterior cells of the Drosophila wing to secrete other signaling molecules such as Wg or Dpp (Chen and Struhl, 1996).

It has been proposed that the protein Patched (Ptc), a multiple-pass transmembrane protein composed of channels and transporters, acts as the Hh receptor (Nusse, 1996). The receptor has two properties: to bind and sequester Hh (thus preventing the spread of Hh from posterior to anterior compartments) as well as to cause changes in cellular behavior such as transcription of various genes as a result of binding (Chen and Struhl, 1996). The ptc gene is located downstream of hh in Drosophila and acts upstream of other genes in the pathway encoding signaling components involved in the activation of wg and dpp; i.e., fused (fu) and cubitus interruptus (ci) (Marigo et al., 1996). Although Hh is required for Wg and Dpp expression, its main function is to repress the ptc gene (Nusse, 1996). This repression results in the regulation of the spatial expression of wg, dpp and ptc. Further analysis of ptc gene interaction indicates that ptc represses the expression of wg and dpp (hh is required for wg and dpp expression), revealing that in the absence of functional Ptc, wg and dpp are ectopically expressed. Furthermore, Ptc represses its own function leading to high expression of inactivated Ptc. The upregulation of ptc by Hh reflects a novel, "self-stimulating" mechanism by which Hh restricts its own range of action. A hh;ptc double mutant resembles the ptc single-mutant phenotype, revealing that Hh is not necessary in the absence of ptc and further illustrates that the suppression of ptc is the main function of Hh.

Experiments that demonstrate the involvement of ptc in Hh signaling involved transfecting mammalian cells with ptc or injecting ptc mRNA into Xenopus oocytes (Nusse, 1996). Results indicated that labeled Hh protein binds with high affinity to ptc-expressing cells. Furthermore Hh and Ptc can be co-immunoprecipitated and crosslinked, giving further evidence that Ptc is the Hh receptor. Additional experimental evidence indicated that clones of cells that lacked ptc became permeable to the Hh signal. Thus, the general conclusion of these experiments illustrates that ptc acts as the primary receptor of Hh as well as a sink, functioning to soak up the available Hh signal. A further experiment involving the vertebrate Hh homolog, Shh, revealed the domains of the Ptc receptor required for Shh interaction (Marigo et al., 1996). The Ptc protein, spanning the membrane twelve times, displays a symmetrical conformation revealed by two large extracellular loops. Through mutational deletions of the loops, it was confirmed that both of the extracellular loops were required in Shh binding to Ptc, either by direct contact or for eliciting the correct conformation of the binding site.

As indicated above, ptc is also repressed by the gene smo. Ptc may not signal directly into the cell, but rather interact with Smo, another membrane-bound protein located downstream of ptc in Hh signaling (Marigo et al., 1996). The gene smo is a segment-polarity gene required for the correct patterning of every segment in Drosophila (Alcedo et al., 1996). The smoothened gene encodes an integral membrane protein with characteristics of heterotrimeric G protein-coupled receptors; i.e., 7- transmembrane regions. This protein shows homology to the Drosophila Frizzled (Fz) protein, a member of the wingless pathway. It was originally thought that smo encodes a receptor of the Hh signal. However, this suggestion was subsequently disproved as evidence for Ptc being the Hh receptor was obtained. Cells that express Smo fail to bind Hh, indicating that Smo does not interact directly with Hh (Nusse, 1996).

Smo is required by anterior cells to transduce Hh and to impede the spread of Hh into the anterior compartment (Chen and Struhl, 1996). Smo mutants display a similar phenotype to Hh mutants. Loss of Smo activity prevents the ability of anterior cells to respond to Hh. As a result, Hh will spread into the anterior compartment until it reaches cells expressing smo. Smo-mutant cells behave as if they cannot transduce Hh, and this leads to a phenotype in which the anterior compartment develops as a symmetrical bifurcation or double anterior winglet.

It was observed, through co-immunoprecipitation, that when Smo and Ptc were coexpressed, a complex formed between the two proteins (Nusse, 1996). A heteromeric-signaling complex may be formed between Smo and Ptc, whereby Ptc binds the Hh ligand initially and signal transduction is carried out by Smo. Further data regarding the interaction between these proteins in Hh signaling have shown that Ptc, in the absence of Hh, inactivates the Smo protein whereby the subsequent binding of Hh to Ptc alleviates the inhibition, thus activating Smo. Smo can therefore activate downstream genes wg, dpp and ptc through various signaling components (Fu, Ci). This event may occur through inhibiting protein kinase A (PKA), yet little evidence supports PKA regulation by Hh signaling.

Even though Smo is not the receptor for Hh, as was originally hypothesized, its role in the transduction of the Hh signal remains prevalent. As the primary receptor for Hh, Ptc binds the Hh ligand, subsequently affecting the transcrption of downstream genes. Given this basic collaboration between Hh and the components of its signaling pathway, much more research must be done in order to determine the exact mechanisms underlying specific genetic and molecular interactions.



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References

Alcedo, J., et al. 1996. The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the Hedgehog signal. Cell. 86: 221-232.

Chen, Y., and Struhl, G. 1996. Dual roles for Patched in sequestering and transducing Hedgehog. Cell. 87: 553-563.

Marigo, V., et al. 1996. Biochemical evidence that Patched is the Hedgehog receptor. Nature. 384: 177-179.

Nusse, R. 1996. Patching Up Hedgehog. Nature. 384: 119-120.

Stone, D.M., et al. 1996. The tumor-suppressor gene patched encodes a candidate receptor for Sonic Hedgehog. Nature. 384: 129-133.



Jeff Wakeford, Pauline Li, Lisa Ricketts and Jennifer Brandon. 1997. The Role of Patched and Smoothened in Hedgehog Signaling. In L.W. Browder (Ed.), Developmental Biology, <http://www.ucalgary.ca/~browder>.

Copyright © 1997 Jeff Wakeford, Pauline Li, Lisa Ricketts and Jennifer Brandon. This material may be reproduced for educational purposes only provided credit is given to the original source.

December 11, 1997