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Hedgehog (HH) proteins are now well established as one of the principal families of secreted signals that orchestrate the development of both invertebrate and vertebrate embryosl. Although much has been learned in recent years about the diverse roles of these proteins, our understanding of the pathways that trans- duce their activities remains fragmentary. The dis- covery earlier this year that the human homologue of a putative HH receptor-encoded by theDrosophila segment-polarity gene patched - is a tumour sup- pressor associated with basal cell carcinoma2,” has added a new dimension to the interest in this path- way. Now, two further papers4r5 have reported the identification of an alternative candidate for a HH receptor. Intriguingly, this protein, encoded by the smoothened gene, exhibits limited homology to mem- bers of the Frizzled family of serpentine proteins, themselves recently implicated as receptors for another major family of secreted signals, the Wnt proteins6.

HEDGEHOGS - secreted proteins with novel properties

All members of the HH family-which include the colourfully named SONIC, INDIAN, DESERT, TIGGY- WINKLE and ECHIDNA - have a highly conserved N-terminal region containing a signal sequence and a much more divergent C-terminal region. Initial stud- ies revealed that these two halves are liberated by an autoproteolytic cleavage reaction catalysed by a con- served domain within the C-terminal fragment’s. Since all of the signalling activity of the proteins re- sides within the N-terminal fragment, the only func- tion of the C-terminus appears to be promoting the autoproteolysis. So, what is the functional significance of this processing mechanism? A striking character- istic of HH proteins is that, with few exceptions, they act as short-range signals, acting directly over, at most, a few cell diametersr. Consistent with this, when the proteins are expressed in tissue-culture cells, most of the N-terminal fragment ends up associated with the cell membrane. By contrast, when truncated cDNAs encoding the mature N-terminal fragment of the pro- tein are expressed similarly, the same fragment is found predominantly in the medium, not stuck to the cell surface. Beachy and colleagues9 have now shown that the autoproteolysis reaction itself results in an increase in hydrophobicity of the signalling fragment, probably through the addition of some novel lipophilic moiety to its C-terminal end. Interest- ingly, this modification influences the subcellular distribution of the fragment - normally, HH is local- ized to the basolateral region of epithelial cells in Drosophila embryos [in contrast to proteins anchored by glycosylphosphatidylinositol (GPI)], whereas ex- pression of the unmodified protein shows an apical distribution. It seems unlikely that this specific local- ization is a prerequisite for interaction with its recep- tor since the unmodified N-terminal fragment is equally effective in activating HH target gene@.

PATCHED - a suppressor of the HEDGEHOG response One fortuitous outcome of the analyses of HH pro-

cessing has been the development of protocols for the efficient production of biologically active soluble

trends in CELL BIOLOGY (Vol. 6) December 1996

hedgehogs

Studies of the HEDGEHOG signallingpathway in Drosophila have

revealed a fimctional link between two genes, cubitus interruptus

and patched, whose human homologues are, respectively, a

proto-oncogene and a tumour suppressor. While the former has

been implicated as a transcription factor, controversy has

surrounded the function of the transmembrane protein encoded by

the latter. Somewhere in the signal-transduction pathway between

these two lies protein kinase A (PKA), and now SMOOTHENED,

whose similarity to G-protein-coupled receptors suggests a link with

PICA, has also been implicated in the pathway. This article

summarizes the current understanding of the pathway and the

interactions between these proteins.

HH protein. Despite this ready source of protein, there have, as yet, been no reports of HH-binding proteins being identified by biochemical approaches. Instead, identification of putative HH receptors has relied upon genetic analysis; and until recently the only candidate identified in this waylo has been the novel multipass transmembrane protein encoded by putched (ptc) r1J2. Unusually for a receptor, inactiva- tion of PTC results in the de-repression of genes - such as wingless (wg) and decapentaplegic (dpp) -whose expression is normally induced by HH; and crucially, the expression of these genes becomes independent of HH activity in the absence of PTC function. Since PTC is normally expressed both in cells that are ac- tively responding to secreted HH and in cells in which the HH pathway is inactive, these genetic data sug- gest a simple model (Fig. la), whereby HH acts by antagonizing the activity of PTC (Ref. 10). And since PTC is a transmembrane protein, it seems at least plausible that this antagonism may be accomplished by a direct interaction between the two proteins. How- ever, doubts have lingered over this model, not least because the predicted topology of PTC proteins is not typical of well-characterized receptors, resembling more that of channels or transporter proteinsr1J2. Moreover, the phenotype of ptc-hh double mutant embryos, although similar, is not identical to that caused by removal of PTC activity aloner3. So, al- though HH is not needed to activate wg or dpp when

0 1996 Elsevier Science Ltd

The authors are in the Molecular Embryology Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn

Fields, London, UK WCZA 3PX. E-mail: ingham@ icrf.icnet.uk

451 PII: SO962-8924(96)30072-X

m FORUM

(a) (W

ptc is absent, it appears to be necessary for some other process that is independent of ptc.

SMOOTHENED - a more likely suspect? Given these caveats, the possibility that reception of

the HH signal is mediated by a protein other than PTC did not seem too unlikely. Accordingly, the dis- covery that the smoothened (smo) gene, whose activity is essential for HH signalling, encodes a protein pre- dicted to contain seven-transmembrane domains4J - a topology similar to that of members of the large family of G-protein-coupled receptors (GPCRs) -seems at first sight to present an attractive alternative to PTC. Paradoxically, however, genetic analysis indicates that smo acts downstream of ptc (Ref. 4), which is not the anticipated relationship if SMO itself acts as the HH receptor. Of course, it is quite possible that PTC and SMO act in parallel pathways. Yet the fact that HH targets are expressed in ptc-hh mutant embryos

FIGURE 1

The growing complexity of the HEDGEHOG (HH) pathway. (a) The genetic analysis of hh and patched (ptc) mutants

together with the molecular characterization of PTC suggested a simple model whereby PTC activity represses the transcription of HH target genes. Activation of these targets would occur only when PTC activity is antagonized by HH, presumably by a direct interaction between the two proteins at the cell surface. (b) The discovery that smoothened (smo) encodes a seven-transmembrane protein required for HH signalling introduces a further complexity into the model. At first sight, an attractive model is that HH interacts directly with SMO. However, since PTC acts upstream of SMO, such an interaction is by no means a foregone conclusion. The most that can be deduced from the genetic data is that PTC negatively regulates SMO and that HH overcomes this inhibition somehow, either by binding to SMO or to PTC (or indeed to a complex of the two transmembrane proteins). (c) Once transduced across the cell membrane, the HH signal acts apparently by increasing the intracellular levels of the transcription factor CUBITUS INTERRUPTUS (Cl), which in turn activates transcription of target genes including decapentaplegic (dpp) and wingless (wg). Protein kinase A (PKA) opposes this effect of HH, keeping intracellular levels of Cl low. How these two opposite influences interact is unclear, but it seems likely that the FUSED serine/threonine kinase, which is itself phosphorylated in response to HH, plays an important role.

but not in smo-ptc double mutants implies that, in the absence of PTC, SMO itself is active independently of its putative ligand - and it follows from this that a direct interaction between HH and SMO is not essen- tial for the activity of the latter. In this light, models that invoke a direct interaction between PTC and SMO that is antagonized by HH (either by binding to SMO or to PTC or to a complex of the two) seem equally plausible (see Fig. lb). With the genes en- coding all three proteins now cloned, it should be possible at last to apply biochemical techniques to investigate these interactions directly (see Box 1).

Inside the cell Last year, several laboratories reported the surpris-

ing finding that elimination of protein kinase A (PKA) activity in Drosophila results in phenotypes very similar to those caused by ptc loss-of-function mutations14. Thus, cells lacking PKA behave as though

452 trends in CELL BIOLOGY (Vol. 6) December 1996

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they have received the HH signal, implying that the signal may be transduced via the modulation of PKA activity. Similar effects of PKA have been observed subsequently in various vertebrate systems, indicating that this mechanism has been highly conserved through evolution15-17. Given the classic role of GPCRs in regulating intracellular CAMP levels14, this link between a novel GPCR-like protein, SMO, and the CAMP-dependent kinase, PKA, is intriguing. So, does SMO act by modulating CAMP levels? Again, this question has not been addressed at the biochemical level; however, two lines of evidence suggest that the relationship between SMO and PKA is unlikely to be direct: first, a constitutively active form of the PKA catalytic subunit is unable to suppress the HH response (except when expressed at very high levels) in trans- genie DrosophiZa14; and, second, experiments using tissue-culture cells have shown that another protein kinase, encoded by the Drosophila gene @sea, itself essential for HH signalling (at least in the embryo), is phosphorylated in response to HH, a process that is independent of PKA activity18. Taken together, these findings indicate that inactivation of PKA is not necessary for transduction of the HH signal; instead, it seems more likely that the HH pathway and PKA act in opposing ways to modulate the activity of a common target (see Fig. lc).

Irrespective of how PKA might influence CI func- tion, one obvious way in which the HH signal could counteract its effects is by dephosphorylation of PKA target sites. To date, neither are there data that ad- dress the phosphorylation state of CI either in vivo or in vitro, nor have any phosphatases been implicated in the transduction of the HH signal. Clearly, many aspects of this fascinating pathway remain to be un- covered - the continuing exploitation of Drosophila genetics combined with the increased use of bio- chemical approaches will, no doubt, shed further light on it.

A likely candidate for this common target is the product of the cubitus interruptus (ci) gene, a Zr?+-finger protein homologous to the vertebrate GLI family of transcription factors. Loss of CI activity results in loss of HH target expression*, strongly suggesting that CI plays a central role in mediating the nuclear response to HH. One problem with this model is the difficulty in demonstrating convincing levels of CI protein in the nuclei of cells actively responding to HH signal- linglg. Nevertheless, recent studies have provided compelling -evidence that CI does indeed activate transcription by binding to discrete sites upstream of HH target genes 20. Moreover, these studies indicate that simply increasing the intracellular levels of CI protein is sufficient to activate HH target gene ex- pression, even in the absence of HH activityzoJ1. Sig- nificantly, cells responding to HH signalling during normal development show just such an increase in their levels of CI protein (although the levels of ci transcript remain unaffected); and the same effect is observed when PKA activity is eliminated from cellsz2. These observations suggest that the principal role of PKA may be to promote the turnover of CI - HH would then act by reversing this effect, leading to increased levels of CI and hence to the activation of the appropriate target genes. Consistent with this model, the CI protein contains several potential PK.4 phosphorylation sites. Of course, it is always possible that phosphorylation of these sites has some other consequence, perhaps inactivating the protein or inhibiting its nuclear import. Intriguingly, the activity of CI is required in some contexts even in cells that do not receive the HH signal. Thus, in the anterior halves of the developing appendages of the fly, CI is expressed at levels apparently too low to activate HH target genes but nevertheless essential to repress transcription of the hh gene itselfzl.

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,i :, ( I( I' _I" : I','.l',

(. ,_,:_ \ d Box1 _< ,_ _" * " * Ii '.': :) : 2 i)~,‘, I Noteadd~~nproof, " .: c :': :f,,, '~:.:';"";'i~

Two groups@’ have now shown that the vertet&e SONi$ HEDGEHOG protein can indeed bind to PTC.,Moteover$ Rosenthal and colleagues find no evidence for a,siniilar interaction with SMO, but do find that PTC and SMO ?a& be co-irnmunoprecipitated. Taken together, ihese data support the notion that SMQ is inhibited by a direct inter- action with PTC and that this inhibition is+eleased by HH binding to WC. ,, ( .:

aMARfCO, V. et ai. (I 996) Nature (in press} ,: bROSENTllAL, A. et 01. (l996) Nature (in pressj

trends in CELL BIOLOGY (Vol. 6) December 1996 453