STAR

Star interacts with Drosophila EGF receptor in the eye. Mosaic analysis of Star in the larval eye disc reveals that homozygous Star patches contain no developing R cells. Taken together with the expression pattern at the morphogenetic furrow, these results demonstrate an early role for Star in photoreceptor development. Loss-of-function mutations in Star act as suppressors of R7 development in a sensitized genetic background involving the Son of sevenless locus; overexpression of Star enhances R7 development in this genetic background. Based on the genetic interactions with Sos, it has been suggested that Star also has a later role in photoreceptor development including the recruitment of the R7 cell through the sevenless pathway (Kolodkin, 1994).

Protein Interactions

Spatially restricted processing of Spitz may be responsible for DER (EGF-R) graded activation. On the basis of genetic interactions, it has been suggested that the Rhomboid (Rho) and Star proteins act as modulators of EGF-R signaling. No alteration in EGF-R autophosphorylation or the pattern of MAP kinase activation by secreted Spitz is observed when the Rho and Star proteins are coexpressed with EGF-R in S2 cells. In embryos mutant for rho or Star the ventralizing effect of secreted Spitz is epistatic, suggesting that RHO and Star may normally facilitate processing of the Spitz precursor (Schweitzer, 1995).

Activation of the Drosophila epidermal growth factor receptor (Egfr) by the transmembrane ligand, Spitz (Spi), requires two additional transmembrane proteins: Rhomboid and Star. Genetic evidence suggests that Rhomboid and Star facilitate Egfr signaling by processing membrane-bound Spi (mSpi) to an active, soluble form. To test this model, an assay based on Xenopus animal cap explants was used in which Spi activation of Egfr is both Rhomboid and Star dependent. Spi is on the cell surface but is kept in an inactive state by its cytoplasmic and transmembrane domains; Rhomboid and Star relieve this inhibition, allowing Spi to signal. Spi is likely to be cleaved within its transmembrane domain. However, a mutant form of mSpi that is not cleaved still signals to Egfr in a Rhomboid and Star-dependent manner. These results suggest strongly that Rhomboid and Star act primarily to present an active form of Spi to Egfr, leading secondarily to the processing of Spi into a secreted form (Bang, 2000).

Rhomboid and Star-mediated Egfr signaling was analyzed by an assay in which Xenopus animal cap explants were isolated from embryos injected with in vitro synthesized Egfr, spi, rhomboid, and Star mRNA. This assay is based on the fact that Egfr activates the Ras pathway, which should lead to an up-regulation in the expression of the Ras target gene Xenopus Brachyury (XBra) in animal caps. Animal caps from injected embryos were allowed to develop until sibling embryos were late gastrulae (stage 11.5), when they were analyzed for XBra expression by RNAse protection assay (RPA). Expression of XBra can be induced in animal caps by Egfr but only under the same conditions that are required for the activation of Egfr in Drosophila. Thus, expression of XBra is not induced in animal caps that express Egfr alone, Egfr along with mSpi, or Egfr along with just Rhomboid and Star. In contrast, a high level of XBra expression is induced when animal caps express Egfr along with mSpi, Rhomboid, and Star. The requirement for Rhomboid and Star for Egfr activation can be overcome in the animal cap assay, as in Drosophila, by expressing sSpi, an engineered form of Spi that contains just the extracellular domain. In addition, Egfr activation can be blocked, as in Drosophila, by introducing the Egfr inhibitor, Argos (Bang, 2000).

By themselves, Rhomboid and Star each weakly promote mSpi activation of Egfr, however, together they are strongly synergistic. Thus, both Rhomboid and Star may be required to achieve maximal levels of Egfr activation, but for lower levels of signaling, either one alone may be sufficient. It is possible that Rhomboid and Star are obligate cofactors but that there are homologous proteins present in the animal cap that fulfill the role of the missing component, albeit weakly. Alternatively, this result may reflect a way in which various levels of receptor activation may be achieved. In some settings, such as the Drosophila wing veins, rhomboid and Star are codependent, whereas in the eye, Star is sufficient and rhomboid function appears to be dispensible (Bang, 2000).

Next, a determination was made whether Rhomboid and Star are required for Egfr activity by acting in the signaling cell, the receiving cell, or in both cells. To do this, activation of XBra was measured in sandwiches that were made by combining an animal cap expressing Egfr with another animal cap expressing mSpi, in the presence or absence of Rhomboid and Star. When Rhomboid and Star are present in the receptor-expressing cells, mSpi fails to activate Egfr. However, when Rhomboid and Star are present in the ligand-expressing cells, mSpi strongly activates Egfr. It has been suggested that Rhomboid and Star may act as cell adhesion molecules to bring together the receptor and ligand into a cell surface complex. To test this idea, sandwiches were made in which rhomboid and Star were expressed in both the sending and receiving cells. Interestingly, this configuration attenuates the level of Egfr signaling, with the strongest repression occuring when both Rhomboid and Star are present on both sides of the sandwich. It is an intriguing possibility that an interaction between Rhomboid and/or Star in trans may dampen the level of signal received by Egfr, providing another possible mechanism by which the level of Egfr activation could be finely tuned. Together, these results argue against models in which Rhomboid and Star regulate receptor function or act as cell adhesion molecules and support a model in which Rhomboid and Star potentiate Egfr activation by acting in the signaling cell (Bang, 2000).

It was asked whether Rhomboid and Star potentiate Egfr signaling by changing the activity of its ligand, as suggested by the observation that sSpi does not require Rhomboid and Star to activate Egfr, whereas mSpi does. To address this question, a series of chimeras were made by replacing portions of human TGF-alpha, a vertebrate homolog of Spi, with the corresponding regions from mSpi. Human TGF-alpha alone strongly activates the human EGFR in the animal cap assay. Strikingly, when the cytoplasmic (C) and transmembrane (TM) domains of TGF-alpha are replaced with those of mSpi (TGF-alpha/SpiTMC), the chimeric molecule activates the human EGFR only when Rhomboid and Star are present. In contrast, chimeric molecules in which the TGF-alpha C or TM domains are replaced separately with those of mSpi (TGF-alpha/SpiC and TGF-alpha/SpiTM, respectively) are constitutively active. Thus, together the mSpi TM and C domains are sufficient to confer Rhomboid and Star dependence on TGF-alpha. This result suggests that the C and TM domains maintain Spi in an inactive state, and that their ability to do so is transferrable to another EGFR ligand. As predicted by this interpretation, a membrane-bound form of Spi that activates Egfr signaling in the absence of Rhomboid and Star can be generated by replacing the mSpi TM and C domains with those of TGF-alpha (Spi/TGF-alphaTMC). In addition, SpiDelta53C, a Spi mutant in which 53 carboxy-terminal residues are deleted and 17 cytoplasmic residues remain, exhibits some Rhomboid and Star-independent activity, providing further evidence that the C domain plays an inhibitory role. Together these results argue strongly that the C and TM domains of mSpi act to maintain an inactive state, with ligand activation occuring upon interaction with Rhomboid and Star (Bang, 2000).

One way in which Rhomboid and Star could lead to ligand activation is by promoting proteolytic processing, thus converting mSpi into a form similar to sSpi. Given the possibility that only low levels of Spi are required to activate Egfr, a more sensitive assay was used to determine whether Rhomboid and Star promote proteolysis of Spi. Conditioned medium was prepared from dissociated animal cap cells from embryos injected with RNA encoding sSpi, or mSpi, or coinjected with RNAs encoding mSpi, Rhomboid, and Star. Egfr-injected animal caps were incubated in the conditioned medium and then analyzed for expression of XBra. The conditioned medium from animal caps expressing sSpi or mSpi/Rhomboid/Star contains an activity that activates Egfr, whereas that from animal caps expressing mSpi alone does not. In addition, the conditioned medium activity is Egfr dependent, as it is ineffective on uninjected animal caps. These results suggest that Rhomboid and Star activate mSpi by promoting its cleavage and secretion (Bang, 2000).

Next, it was determined whether proteolytic processing is required for Rhomboid and Star activation of mSpi. To do this, potential sites for processing of mSpi were removed by deleting the sequences encoding the 15 amino acids (aa) between the Spi EGF and TM domains (Spi-15aa). This region was selected because cleavage of TGF-alpha is known to take place within an analogous interval. When tested in the animal cap assay, Spi-15aa strongly activates Egfr in a Rhomboid and Star-dependent manner. In contrast, conditioned medium prepared from animal caps expressing Spi-15aa, Rhomboid, and Star does not contain any activity that activates Egfr, indicating that Spi-15aa is not cleaved. Taken together, these results suggest that cleavage of mSpi depends on the sequence deleted in the Spi-15aa mutant; however, mSpi does not need to be cleaved to activate Egfr signaling. Thus, Rhomboid and Star may act to present mSpi to Egfr and subsequently facilitate or allow its cleavage (Bang, 2000).

The results obtained with the Spi-15aa deletion mutant suggest that mSpi, like TGF-alpha, is processed to generate a soluble form. To examine the nature of this processing further, the possibility was tested that the processing includes a cleavage within the transmembrane domain of mSpi. This possibility is suggested by the results obtained with the Spi/TGF-alpha chimeras, showing that the Spi transmembrane domain is important for Rhomboid and Star-dependent activation. Moreover, another multimembrane-spanning protein, Presenilin-1, mediates proteolyis of the beta-amyloid precursor protein and Notch, both of which are cleaved within their transmembrane domains. If processing does lead to a cleavage in the membrane, it was reasoned that this would release the intracellular domain of Spitz in a Rhomboid/Star-dependent manner. To detect this cleavage, a chimeric molecule was generated in which the mSpi C domain is replaced with the myc-tagged, intracellular domain of the Xenopus Notch receptor (Spi/NICD). The endogenous, gamma-secretase-dependent Notch cleavage site is not present in the Spi/NICD chimeric molecule. If proteolytic processing of this molecule occurs within the Spi TM domain in a Rhomboid/Star-dependent manner, NICD may be released, translocate to the nucleus, and activate target genes. As a Notch target gene Xenopus Enhancer-of-split-related-1 (Esr-1) was analyzed in animal caps that were coinjected with the neuralizing factor noggin, because Esr-1 is normally expressed in neural tissue and its induction by NICD is more robust in a noggin background (Bang, 2000).

When tested in the animal cap assay, Spi/NICD activates Egfr, but only in the presence of Rhomboid and Star, indicating that the Spi/NICD chimeric molecule still exhibits Rhomboid and Star-dependent Spi activity. This result also indicates that the myc-tagged Xenopus NICD can effectively replace the Spi C domain, suggesting that the ability of the C domain to maintain Spi in an inactive state depends more on its structure than on its primary sequence. Significantly, Spi/NICD also activates the Notch target gene, Esr-1, in a Rhomboid and Star-dependent manner. This result suggests that Rhomboid and Star promote a proteolytic processing event within the Spi-TM domain that releases NICD. In addition, because Esr-1 induction is Rhomboid and Star dependent in the absence of Egfr, Rhomboid and Star can function independent of Egfr (Bang, 2000).

By analogy to the beta-amyloid precursor protein and Notch, whose activites are regulated by multiple, interdependent cleavage events, the possibility was tested that the 15 amino acids between the Spi EGF and TM domains that are required for production of soluble Spi are also required for the cleavage of the Spi/NICD chimeric molecule within its TM domain. Thus, the sequence encoding these 15 amino acids was depleted in the Spi/NICD chimera to produce Spi-15aa/NICD. This deletion mutant still strongly activates Egfr in a Rhomboid and Star-dependent manner, but no longer induces Esr-1, indicating that NICD is not released, and thus cleavage of this mutant does not occur. Thus, these results provide further independent evidence for the contention that Rhomboid and Star-dependent cleavage of mSpi requires the amino acids deleted in the Spi-15aa mutant, but mSpi need not be cleaved to activate Egfr signaling. Finally, these results suggest that there is a Rhomboid and Star-dependent cleavage event of mSpi within its TM domain. One possible explanation for these observations is that mSpi is cleaved both within the TM domain and within the 15 amino acids between the TM and EGF domains. Alternatively, a single cleavage of mSpi could occur within its TM domain that depends on the 15 amino acid interval (Bang, 2000).

Several models could account for the Rhomboid and Star-dependent effects observed. One model is that Rhomboid and Star are required to direct mSpi to the proper compartment for signaling to occur. The results from the biotinylation experiments suggest strongly that Rhomboid and Star are not required for transport of mSpi to the cell surface, but it remains a possibility that Rhomboid and Star could play a role in localizing mSpi to specific cell surface microdomains. An alternative class of models is that mSpi is at the cell surface and ready to signal, but that Rhomboid and Star are required for bringing mSpi into an active conformation. One version of this model is that Rhomboid and Star activate mSpi by promoting its oligomerization. However, this idea is difficult to reconcile with the observation that sSpi is active and either does not require oligomerization or oligomerizes independently of Rhomboid and Star. In addition, soluble EGF, which is similar to sSpi, binds as a monomer to the extracellular domain of the EGFR in a 1:1 ratio, suggesting that membrane-bound EGFR ligands may also bind the receptor as monomers. For these reasons, an alternative model is favored in which mSpi is present at the membrane in an inactive dimeric or oligomeric complex. Rhomboid and Star would be required to either prevent formation of this complex or to alter its conformation such that mSpi could be presented as an active form. This model is precedented by observations suggesting that a number of receptor tyrosine kinases exist as inactive dimers that are activated when specific inter-subunit conformational changes occur upon ligand binding. Thus, formation of an inactive mSpi complex would be mediated by its C and TM domains and inhibited by an interaction between these domains and Rhomboid and Star. This model explains both why removal of these domains relieves the requirement for Rhomboid and Star, and transfer of these domains to TGF-alpha confers Rhomboid and Star dependence. Such a model also predicts that sSpi would be Rhomboid and Star independent (Bang, 2000).

How do Rhomboid and Star promote cleavage of mSpi? Rhomboid and/or Star could play a passive role by making mSpi accessible to proteolysis upon presentation. Alternatively, Rhomboid and/or Star may actively facilitate Spi proteolysis either by activating or recruiting a protease or transporting Spi to the appropriate subcellular compartment. It is also possible that Rhomboid and/or Star could themselves have proteolytic activity, as has been proposed for Presenilin-1. A protease responsible for Spi cleavage has yet to be identified. Finally, although this study strongly suggests that presentation of Spi is inhibited by its C-domain, the question of whether proteolysis of Spi is also affected by the C-domain has not been addressed. For instance, proteolytic release of the extracellular domain of membrane bound neuregulin is dependent on its cytoplasmic domain. Future experiments will be aimed at determining whether Rhomboid and Star play a passive or an active role in the proteolysis of mSpi (Bang, 2000).

Drosophila Spitz is an activating ligand for the EGF receptor (Egfr). It has been shown that Star is required for Spitz activity. Star is quantitatively limiting for Spitz production during eye development. Star and Spitz proteins colocalize in Spitz sending cells and this association is not coincident with the site of translation, consistent with a function for Star in either Spitz processing or transmission. Minimal sequences within both Spitz and Star have been defined that mediate a direct interaction, and this binding can occur in vivo (Hsiung, 2001).

Genetic analysis has indicated that Star (with rhomboid) may function upstream of the transmission of Spitz from sending cells and Star protein has been shown to be localized to the nuclear envelope and the early ER. Taken together, these data suggest that Star may function in the translation, post-translational cleavage, glycosylation, secretion or presentation of Spitz. To approach this, Star and Spitz proteins and spitz mRNA were localized in a series of pair-wise double stains at the confocal level. Spitz and Star proteins do colocalize, but spitz mRNA does not. Furthermore Spitz and Star proteins appear together in granular structures that are perinuclear as well as apical in the cells. This suggests that the Spitz-Star interaction persists through much or all of the secretory pathway (Hsiung, 2001).

Controlling the quantity of Star directly affects the quantity of Spitz antigen seen (in loss- and gain-of-function mosaic clones and by ectopic expression in the entire eye). In short, less Star results in less Spitz and more Star in more (and ectopic) Spitz. Consistent with this, it was found that overexpression of unprocessed full length mSpitz alone has no phenotypic effect, but overexpression of Star does result in a moderate rough eye suggesting that normally spitz RNA is in excess and the quantity of the signal is limited by Star. Furthermore, overexpressing both mSpitz and Star together results in a synergistic effect and a grossly disordered eye, with a large excess of photoreceptors and a deficit of accessory cells. Since the main function of the Spitz/Egfr signal in the eye is to recruit cells to the developing clusters and specify them as photoreceptor neurons, this phenotype is consistent with a great increase in the quantity of this signal. Immuno-colocalization data appear to suggest that there is more Star antigen than Spitz in the developing eye. However, it is very difficult to draw any conclusions as to the actual relative abundance of these proteins : these experiments were not quantitative. Taken together, all these data suggest that the quantity of Star protein is the critical limiting factor for the Spitz/Egfr signal, at least during the normal development of the compound eye (Hsiung, 2001).

A series of GST-mediated in vitro binding experiments was used to define a single region, each in Spitz and Star, that mediates their direct interaction with one another. In Spitz, this 48-residue segment (SpitzH) is virtually identical to the 'factor' domain -- that part of the protein that contains the six cysteine residues and other features that show homology to the small diffusible growth factors of the TGF-alpha family. In Star, a 19-amino-acid GFBD responsible for binding to Spitz has been identified. To confirm these results, a test in vivo was conducted: SpitzH was fused to CFP as well as a dominant nuclear localization signal (NLS). This CFP-SpitzH-NLS fusion when expressed in HeLa cells is directed to the nucleus by virtue of the NLS and a cyan-colored signal is detected there by confocal microscopy. A fusion protein was also made in which an evenly distributed yellow fluorescent protein protein kinase (YFP-PK) protein was fused to the GFBD from Star (YFP-Star7-PK). When YFP-Star7-PK is expressed alone in HeLa cells, the yellow signal is evenly distributed, but when coexpressed with CFP-SpitzH-NLS, the yellow signal moves to the nucleus. This 'cargo' experiment confirms that the Spitz factor domain and the Star GFBD can bind in vivo. Taken together, the in vitro 'GST-pull down' experiments and the in vivo HeLa cell 'cargo' experiments are consistent with a direct interaction in the living fly between the Spitz factor domain and the Star GFBB. However, neither of these two experiments tested this interaction in the secretory pathway (Hsiung, 2001).

It is interesting to note that the Spitz 'factor' domain is N-terminal to the Spitz trans-membrane domain, and thus presumed to lie outside of the plasma membrane (or in the lumen of the organelles of the secretory pathway). The GFBD in Star lies C-terminal to its trans-membrane domain and thus would appear to lie on the wrong side of the plasma or organelle membranes to interact directly with the Spitz factor domain. However, structural features of Star have led others to suggest that Star is actually a type II integral protein, with its C-terminus outside and its N-terminus inside. This is therefore consistent with a direct interaction between the Spitz factor domain and the Star GFBD in vivo (Hsiung, 2001).

In summary, Spitz and Star proteins associate in living cells in the developing Drosophila compound eye, Star controls the quantity of Spitz signal, and these proteins interact via the factor domain in Spitz and the GFBD in Star. These data are consistent with a role for Star in some stage or stages of Spitz signal production subsequent to its translation. These conclusions are very similar to those reached by others for Rhomboid family proteins. While no firm conclusions can be drawn from these data, it is suggested that Star may be involved in a complex in the secretory pathway that acts in the maturation of Spitz. Star could act before Rhomboid, because Star has been localized early in the pathway and Rhomboid has been localized to the apical microvillae, or, they may act together. There is no evidence to suggest that either Star or Rhomboid are themselves proteases capable of cleaving Spitz: perhaps they recruit one. Alternately Star may act as a chaperone to route the pro-Spitz protein correctly within the secretory pathway or it might be required for the correct folding of Spitz or it may recruit glycosylation enzymes. Indeed, the data suggest that Star can interact with the Spitz factor domain in vitro in conditions in which it may not be correctly folded. In the developing eye, anterior to the furrow, Star appears to be quantitatively limiting on Spitz expression. It may be that Spitz pro-protein that is not correctly routed or cleaved may be unstable (Hsiung, 2001).

While there are several known Rhomboid proteins, Star appears to be unique in the Drosophila genome. While homologs of Rhomboid have been detected in vertebrates, no homolog of Star has been detected outside of Drosophila. Star is essential in Drosophila for the activation of an otherwise inactive growth factor homolog (Spitz). There may be proteins with similar functions in vertebrates, which have conserved structure but which are too far diverged at the primary sequence level to be found with current computer searching algorithms (Hsiung, 2001).

The membrane proteins Star and Rhomboid-1 have been genetically defined as the primary regulators of EGF receptor activation in Drosophila, but an understanding of their molecular mechanisms has remained elusive. Both Star and Rhomboid-1 have been assumed to work at the cell surface to control ligand activation. This study demonstrates that they control receptor signaling by regulating intracellular trafficking and proteolysis of the ligand Spitz. Star is present throughout the secretory pathway and is required to export Spitz from the endoplasmic reticulum to the Golgi apparatus. Rhomboid-1 is localized in the Golgi, where it promotes the cleavage of Spitz. This defines a novel growth factor release mechanism that is distinct from metalloprotease-dependent shedding from the cell surface (Lee, 2001).

In the absence of Star, Spitz is retained in the ER. This explains why the domain of EGF receptor activation is much narrower than the expression pattern of Spitz, and why ectopic expression of full-length Spitz does not activate the receptor. Star, a protein with a single TMD, chaperones Spitz into the Golgi apparatus and the subsequent secretory pathway. The principal interaction between Spitz and Star occurs between the lumenal domains of the two proteins. Two models can be envisaged: Star could specifically block the ER retention signal; alternatively, Star could actively export Spitz from the ER, and in doing so, counteract retention (Lee, 2001).

Drosophila genetics indicate that Star and Rhomboid-1 are both prime regulators of EGF receptor activity: they both appear to be necessary and they cannot replace each other. It has not been possible to separate their functions. The results described here explain their codependency and synergy, and also provide a clear mechanistic distinction between them. An important issue is whether Star is necessary for Rhomboid-1-dependent proteolysis itself, as an enzymatic cofactor. This possibility can be ruled out, based on the Spi:TGFalpha-C chimera: it leaves the ER independently of Star and is efficiently cleaved by Rhomboid-1 in the absence of Star, implying that the primary function of Star is to export Spitz from the ER, thereby allowing it access to Rhomboid-1. Note, however, that the Rhomboid-1-dependent cleavage of the Spi:TGFalpha-TMC chimera suggests a possible secondary, nonessential role for Star as an adaptor, delivering substrate to Rhomboid-1. The data also do not rule out a role for Star in promoting efficient Spitz secretion (Lee, 2001).

The data clearly show that Rhomboid-1 is a Golgi-localized protein that triggers the proteolytic cleavage of Spitz. Rhomboid-1 could therefore be a novel protease, or it could recruit an unidentified protease; detailed biochemical analyses will be needed to resolve this. Star and Rhomboid-1 are sufficient to cleave Spitz in all cells tested, suggesting that they may be the only components required. The analysis also rules out the involvement of metalloproteases that are responsible for the release of TGFalpha and many other growth factors, further supporting the idea that Rhomboid-1 may itself be a protease. The absence of a genetically identified candidate protease, other than Rhomboid-1, despite much genetic screening, is also consistent with this hypothesis. The principal objection to this parsimonious model is Rhomboid-1's lack of identifiable protease domains. However, there are two recent precedents for multiple transmembrane domain proteins without recognizable protease domains being discovered to be novel proteases: presenilin-1 and SREBP site 2 protease (Lee, 2001).

Despite the distinctions between Spitz and TGFalpha processing, the similarities between flies and mammals may be greater than is at first apparent. For example, mature TACE (an ADAM family metalloprotease that acts on TGFalpha) is predominantly localized in intracellular compartments, suggesting that the cell surface may not be the only location of TGFalpha cleavage. Additionally, there is evidence for TACE-independent TGFalpha processing. Furthermore, TGFalpha also undergoes regulated transport through the secretory pathway, albeit by a distinct mechanism dependent on PDZ domain proteins. Finally, it is worth pointing out that while TGFalpha appears to be the mammalian ligand most similar to Spitz, there are several other analagous human EGF receptor ligands whose regulation is still poorly understood (Lee, 2001).

Drosophila has three membrane-tethered epidermal growth factor (EGF)-like proteins: Spitz, Gurken and Keren. Spitz and Gurken have been genetically confirmed to activate the EGF receptor, but Keren is uncharacterized. Spitz is activated by regulated intracellular translocation and cleavage by the transmembrane proteins Star and the protease Rhomboid-1, respectively. Rhomboid-1 is a member of a family of seven similar proteins in Drosophila. Four of the rhomboid family members have been examined: all are proteases that can cleave Spitz, Gurken and Keren, and all activate only EGF receptor signaling in vivo. Star acts as an endoplasmic reticulum (ER) export factor for all three. The importance of this translocation is highlighted by the fact that when Spitz is cleaved by Rhomboids in the ER it cannot be secreted. Keren activates the EGF receptor in vivo, providing strong evidence that it is a true ligand. These data demonstrate that all membrane-tethered EGF ligands in Drosophila are activated by the same strategy of cleavage by Rhomboids, which are ancient and widespread intramembrane proteases. This is distinct from the metalloprotease-induced activation of mammalian EGF-like ligands (Urban, 2002).

Star regulates Spitz cleavage by Rhomboid-1 by transporting Spitz to the Golgi apparatus. Strikingly, although Star was essential for ligand secretion into the culture medium in each case, it does not affect the ability of Rhomboids 2, 3 and 4 to catalyse Spitz cleavage. The extensive O-linked glycosylation that is diagnostic of transit through the Golgi apparatus (and which increases the apparent molecular weight of Spitz) was not present in cell lysates. Therefore, in contrast to Rhomboid-1, Rhomboids 2, 3 and 4 causes the accumulation of an intracellular cleaved Spitz that is not transported past the trans-Golgi network and thus not secreted (Urban, 2002).

Star's role in Spitz activation is to export Spitz from the ER to the Golgi apparatus where it encounters the proteolytic activity of Rhomboid-1. Star also promotes the release of Keren into the medium, suggesting its role is similar to that in Spitz processing. This relocalization of Keren is very similar to the relocalization observed for Spitz, suggesting that Keren also needs to be relocalized to the Golgi apparatus for efficient processing and secretion (Urban, 2002).

Gurken function is restricted to oogenesis where it is required to polarize both major axes of the egg. Recent evidence strongly suggests that Gurken undergoes proteolytic processing in vivo: Gurken is released from the oocyte and is internalized by follicle cells, exists exclusively in a cleaved form in oocytes, and an uncleavable mutant form is inactive. Gurken can be processed directly by Rhomboid proteases 1-4. In the tissue culture assay, Rhomboid protease activity is required for Gurken cleavage and secretion. Although Rhomboid-1 is not required in the female germline, the specific expression of Rhomboid-2 in the early oocyte suggests that a Rhomboid might have a role in Gurken processing. Star can translocate Gurken from the ER to the Golgi apparatus in cell culture and, in some cases, enhance Gurken secretion. Together, these results strongly suggest that Gurken activity, like that of Spitz, is at least partially regulated by Star-dependent ER to Golgi transport. The regulation of Gurken activity, however, also depends on the transmembrane protein Cornichon. Recent evidence in yeast and Drosophila suggests that Cornichon is an ER export factor, raising the question of the relative roles and significance of Star and Cornichon (Urban, 2002).

Spitz (Spi) is the most prominent ligand of the Drosophila EGF receptor. It is produced as an inactive membrane precursor that is retained in the endoplasmic reticulum (ER). To allow cleavage, Star transports Spi to the Golgi, where it undergoes cleavage by Rhomboid. Since some Egfr phenotypes are not mimicked by any of its known activating ligands, an additional ligand (Keren) was identified by database searches. Krn is a functional homolog of Spi since it can rescue the spi mutant phenotype in a Rho- and Star-dependent manner. In contrast to Spi, however, Krn also possesses a Rho/Star-independent ability to undergo low-level cleavage and activate Egfr, as evident both in cell culture and in flies. The difference in basal activity correlates with the cellular localization of the two ligands. While Spi is retained in the ER, the retention of Krn is only partial. Examining Spi/Krn chimeric and deletion constructs implicates the Spi cytoplasmic domain in inhibiting its basal activity. Low-level activity of Krn calls for tightly regulated expression of the Krn precursor (Reich, 2002).

It was of interest to determine whether Karen processing is regulated in a similar manner to Spi. Triggering of Egfr by Spi at stage 10 generates two prominent domains of activation, in the tracheal placodes and ventral ectoderm. These domains correspond to the sites of Rho expression in the tracheal placodes and midline, respectively. dpERK can be readily detected in these domains, while in spi mutant embryos no dpERK is observed at this stage. The capacity of the Krn precursor to rescue spi mutant embryos was examined. When Krn was ubiquitously expressed in the ectoderm of spi^- embryos (using the 69B-Gal4 driver), complete rescue of the dpERK pattern was observed. Induction of the pathway by Krn at the sites of Rho expression in the midline and tracheal placodes indicates that, like Spi, processing of Krn is dependent upon Rho. To test whether Krn cleavage requires Star, Krn was expressed in Star mutant embryos. No rescue of the phenotype was observed, as monitored by dpERK. It is thus concluded that, like Spi, processing of Krn is Rho and Star dependent (Reich, 2002).

A clearer understanding of Spi cleavage has been gained by studies in cells. Efficient cleavage of Spi occurs only in cells in which both Star and Rho are expressed. Spi is retained in the ER through its intracellular domain. Star binds Spi and translocates it from the ER to the Golgi, where Rho functions as a protease and cleaves Spi. Krn-GFP in Drosophila S2 cells shows partial release from retention, manifested by a vesicular distribution, indicating exit from the ER. The functional implication of this was observed through the ectopic phenotypes in various tissues. For instance, whereas ectopic Spi driven by ubiquitous GAL4 in embryos does not prevent hatching of larvae, ectopic Krn leads to lethality. This highlights the importance of retention, since it allows expression of high amounts of Spi protein in the cell, yet controls Spi activity by preventing Spi from reaching further compartments where cleavage occurs (Reich, 2002).

Chimeric and deletion constructs identify the cytoplasmic domains of Spi and Krn as the domains responsible for their different cleavage profiles. This is a result of different levels of retention in the ER. The mechanism of Spi and Krn retention is not yet clear. Spi has also been shown to be retained in a heterologous system of mammalian cells, implicating the action of conserved molecules or an intrinsic property of the protein. In one model, association of the Spi cytoplasmic domain with an additional protein(s) could mediate retention. In that case, it would be expected that Krn would have lower affinity to this protein(s). In another model, the Spi C-terminus itself could have an intrinsic inhibitory capability through protein folding that sterically prohibits association to proteins -- this would carry Spi further in the secretory pathway. In this case, Krn would be expected to possess a higher affinity to such chaperones, that would allow it to exit the ER without total dependence on Star (Reich, 2002).

Compared with Spi or Krn, the cytoplasmic domain of Grk, the third Egfr ligand with a transmembrane domain, is shorter (only 24 amino acids). Deletion of its cytoplasmic domain did not influence signaling by Grk. Like Krn, overexpression of the full-length Grk protein causes ectopic wing phenotypes. It would be interesting to see to what extent Grk is retained in the ER of S2 cells (Reich, 2002).

Expression of Krn in S2 cells allows the mechanism of low-level cleavage, which is Star and Rho independent, to be followed. What is the protease responsible for this cleavage? The sensitivity of Krn cleavage to inhibitors of serine proteases indicates that cleavage may be mediated by a protease of this family. Unlike Rho, which is expressed in a spatially and temporally regulated manner, the protease is expected to be ubiquitously expressed, since ectopic Krn causes abnormal phenotypes wherever it was expressed. This further elaborates the need for tight transcriptional control on Krn expression (Reich, 2002).

Co-expression of Star with Krn in S2 cells raises the amounts of secreted sKrn in the medium. This is a result of the efficient export of Krn from the ER by Star. Since higher levels of cleavage can be obtained by co-expressing both Rho and Star, it would seem to indicate that the protease involved in low-level cleavage is less efficient than Rho (Reich, 2002).

High-level cleavage of Krn was followed in embryos through the detection of dpERK. The activation profile followed the restricted expression of Rho, since Star is broadly expressed. Only in cell culture could Rho enhance cleavage of Krn without co-expression of Star. This probably occurs because Krn can 'leak' out of the ER, reach compartments where Rho is present and undergo cleavage by Rho independent of Star (Reich, 2002).

Sequence conservation within the transmembrane domains of the Rho protein have suggested that the cleavage site of Spi would reside within the transmembrane domain. There is also evidence to indicate that Rho cleaves Spi within the membrane. The transmembrane regions of Spi and Krn are conserved, and show 50% sequence identity. The transmembrane domain of Krn may thus possess the same recognition sites as that of Spi (Reich, 2002).

In conclusion, Krn was found to be the functional homolog of Spi. Unlike Spi, Krn is capable of undergoing inefficient Star- and Rho-independent cleavage in flies and in cell culture. This is due to differences between the intracellular domains of Krn and Spi, which allow Krn to evade retention in the ER and reach further along in the secretory pathway. This calls for tight transcriptional control of Krn expression, in contrast to Spi, which can be ubiquitously and abundantly expressed (Reich, 2002).

Rhomboid and Star and the activation of Gurken

The mechanism of activation of the Epidermal growth factor receptor (Egfr) by the transforming growth factor alpha-like molecule, Gurken (Grk) has been examined. Grk is expressed in the oocyte and activates the Egfr in the surrounding follicle cells during oogenesis. Expression of either a membrane bound form of Grk (mbGrk), or a secreted form of Grk (secGrk), in either the follicle cells or in the germline, activates the Egfr. In tissue culture cells, both forms can bind to the Egfr; however, only the soluble form can trigger Egfr signaling, which is consistent with the observed cleavage of Grk in vivo. The two transmembrane proteins Star (S) and Brho (rhomboid-2) potentiate the activity of mbGrk. These two proteins collaborate to promote an activating proteolytic cleavage and release of Grk. After cleavage, the extracellular domain of Grk is secreted from the oocyte to activate the Egfr in the follicular epithelium (Ghiglione, 2002).

Grk is cleaved in the germline. An important question is where exactly the cleavage of the Grk precursor occurs? Other studies have concluded that the cleavage of Spitz occurs in the TM and depends on the 15 amino acid stretch located between the EGF and TM domains. The Grk dibasic signal (R₂₄₀ and K₂₄₁) is not the cleavage site because its mutation does not abolish this event. However, mbGrkDelta19AA^myc, in which the 19 amino acid (Y₂₂₄ to V₂₄₂) located between the EGF and TM domains have been deleted, is no longer cleaved, suggesting that this sequence is directly or indirectly involved in the processing (Ghiglione, 2002).

The results do not rule out the hypothesis that Grk cleavage occurs in the TM domain as proposed for Spi. The high conservation between the Spi and Grk TM domains, in addition to aberrant Grk localization observed with different grk alleles affecting this TM domain, reveals its importance. Moreover, the cleaved product of Grk that is released in the medium, after co-expressing mbGrk+S+Rho-1/Brho in S2 cells, has a slightly higher mobility that the engineered secGrk. Thus, it is possible that mbGrk is cleaved within the TM domain and that proteolysis depends on the 19 amino acid interval (Ghiglione, 2002).

These results reflect the importance of the Grk TM domain for proper processing and routing through the secretory pathway. mbGrk processing is probably tightly regulated and leads to efficient Grk secretion, contrary to engineered secGrk, which is poorly secreted from the oocyte and which acts mainly intracellularly (Ghiglione, 2002).

The recent findings that Star and Brho, a Rho-related protein, are expressed in the oocyte led to an investigation of whether they are involved in Grk activation during oogenesis. Star and Rho proteins have been proposed to be involved in the processing and activation of Spi; however, because they have no obvious motifs that predict their biochemical functions, their roles in ligand maturation and/or secretion have remained obscure (Ghiglione, 2002).

The analysis of these proteins in the context of Grk signaling has provided numerous insights into the relationships between these transmembrane proteins. The in vivo data strongly suggest that the expression level of Star and Brho is very high in the oocyte, thus leading to an efficient cleavage and secretion of Grk. However, Star and Rho-1 are probably expressed at low level in the follicle cells. Indeed, the presence of Star in this epithelium using an anti-Star antibody has not been detected, whereas they clearly show a strong staining in the germline. The presence of both endogenous Star and Rho-1 in follicle cells explains why overexpression of mbGrk in this epithelium leads to a weak dorsalization of the eggs. Nevertheless removing one copy of Star is sufficient to completely suppress this phenotype. This confirms the observation that overexpression of mbGrk on its own is not able to activate the Egfr in vivo, as supported by the in vitro study. Overexpression experiments in follicle cells indicate a strong synergy between mbGrk, Star, and Brho, as previously observed for Spi. Further, co-expression of Star and Rho-1/Brho is sufficient for Grk cleavage and secretion in S2 cells, strongly suggesting that they are the only proteins required for this process. In addition, these tissue culture experiments reveal that Star and Rho-1/Brho are not obligate cofactors for this cleavage, because co-expression of mbGrk with Rho-1/Brho is sufficient to catalyze this proteolytic event. Star is not required for Rho-1/Brho-mediated proteolytic cleavage in S2 cells, but the soluble Grk extracellular domain is no longer detected in the medium from these cells, indicating that the function of Srar is necessary for trafficking/secretion of the ligand. However, Star is not able to cleave Grk in absence of Rho-1/Brho. Altogether, these results show that the functions of Rho-1/Brho and Star are distinct, which explains their co-dependence and synergism in vivo (Ghiglione, 2002).

Mosaic analysis of Star, both in the germline and in follicle cells, together with the Star antisense experiment, demonstrate that Star is required in follicle cells for Spi-dependent Egfr activation, and in the germline for Grk-dependent Egfr activation. Tissue culture experiments suggest that Star is not involved in Grk proteolysis, but instead in post-cleavage trafficking or secretion of the ligand. The intracellular localization of Star is also consistent with a role for Star at a step that follows the Brho-dependent cleavage, because it was found that Star is predominantly very close to, or at the plasma membrane, while Brho localizes to the Golgi. The role of Star, however, is not yet resolved because the results contrast with the ER localization of Star in the oocyte described by others. Interestingly, unlike Rho-1 and Brho, Star is probably involved in other processes as well. For example, Star has been identified as a suppressor of Delta, one of the Notch ligands. Delta encodes a transmembrane protein that is cleaved by the Kuzbanian metalloprotease, and the extracellular fragment antagonizes the function of the membrane-bound Delta protein as an activating Notch ligand. In the case of Notch signaling, a reduction of Star gene activity might lead to a reduced release of the extracellular Delta fragment, and thus enhance Delta signaling (Ghiglione, 2002 and references therein).

Finally, understanding the function of Star and Rho-1/Brho in Grk processing is relevant to studies of the mammalian ligands of the EGFR family as well, because TGFalpha may also be processed in vivo before receptor binding. Thus, although further work is needed to fully understand the biochemical function of Star, and Rho-1/Brho, these studies have provided a number of insights into the mechanism of action of these molecules (Ghiglione, 2002).

As a first step in assessing the function of brho, the GAL4-UAS system was used to misexpress a UAS-brho construct containing the full genomic sequence of brho in the wing. When this UAS-brho construct was expressed using the strong ubiquitous wing-specific GAL4 driver MS1096, ectopic vein phenotypes were observed similar to, although weaker than, those generated by misexpression of rho. This observation suggests that Brho functions like Rho by promoting Egfr signaling. To test whether the induction of ectopic veins by brho misexpression requires Egfr activity, UAS-brho was co-expressed with a dominant-negative Egfr construct, UAS-DN-Egfr. As previously observed for rho, brho-induced ectopic veins are entirely suppressed by DN-Egfr, resulting in narrow wings with missing veins typical of DN-Egfr misexpression. Strong synergism was observed between misexpressed brho and Star, as has been shown to be the case for rho. These results are consistent with brho functioning to promote Egfr signaling (Guichard, 2000).

Since Egfr signaling results in MAPK activation, the activation state of MAPK was assessed following misexpression of brho in the wing disc. MAPK is an essential downstream component required to transduce signals from all RTKs to the nucleus. Activated MAPK (MAPK*) can be detected in situ, using an antibody directed against phosphorylated MAPK (anti dP-ERK antibodies). In wild-type wing discs, MAPK activation is restricted to vein primordia, as a consequence of endogenous localized rho expression. In wing discs ubiquitously misexpressing brho, a strong general activation of MAPK was observed comparable to that found in discs ectopically expressing rho or an activated form of Egfr. This observation provides independent support for brho activating the Egfr/MAPK signaling pathway (Guichard, 2000).

As a direct measurement of Egfr activity during oogenesis, wild-type ovaries were probed with anti-dP-ERK antibodies. During early stages, MAPK activation is detected only in posterior follicle cells abutting the oocyte in which brho and gurken are expressed. This pattern of MAPK activation is temporally correlated with brho expression and is consistent with the hypothesis that brho participates in promoting Egfr signaling in posterior follicle cells. It is noteworthy that rho, which activates Egfr signaling in many other developmental settings, is not expressed in the oocyte or surrounding follicle cells during this period (Guichard, 2000).

During later stages of oogenesis (9-10), MAPK activation is restricted to follicle cells overlying the dorsal anterior end of the oocyte. This restricted activation of MAPK is believed to be the result of the asymmetrical localization of gurken transcripts to the dorsal anterior portion of the oocyte, which then resolves into a double peak as a consequence of rho, argos, and spitz activity in the dorsalmost anterior follicle cells at stage 11. Interestingly, a trace of posterior activation of MAPK is also observed at stage 10, suggesting that sustained posterior Egfr activity may maintain posterior fates of the egg chamber (Guichard, 2000).

Whether brho could activate the Egfr pathway in the ovary was tested by expressing the UAS-brho construct under the control of the CY2-GAL4 driver, which is expressed only in the follicular epithelium covering the oocyte. This ubiquitous follicle cell expression of brho causes dorsalization of the eggshell, resulting in thickened dorsal appendages which were more spread apart in eggs from CY2;UAS-brho females than in wildtype controls. In some more extreme cases, white appendage-like material filled in between the two appendages, as is typical of dorsalized eggshells. The average brho misexpression phenotype is similar to, but weaker than, that induced by ectopic rho in follicle cells using the same GAL4 driver (Guichard, 2000).

Star is expressed in the oocyte during a developmental window (stage 4 to 7) largely overlapping with brho expression. Since Star and rho act in concert during many stages of development and function in a strict interdependent fashion during wing vein development, tests were performed to see whether brho might also interact synergistically with Star. UAS-brho and UAS-Star constructs were coexpressed during wing development using the strong ubiquitous GAL4 driver MS1096, and highly penetrant pupal lethality was observed. Despite the pupal lethality, fully differentiated wings can be dissected from pupal cases, revealing a strong ectopic vein phenotype which is much greater than that observed in response to ectopic brho alone. Since ectopic expression of Star alone has no detectable effect, this result reveals a potent synergism between Brho and Star in enhancing Egfr activity during wing development (Guichard, 2000).

A strong effect on brho activity was observed from reducing the dose of endogenous Star since brho-induced ectopic veins are almost completely suppressed in a Star2/1 heterozygous background. These results indicate that Star can collaborate with brho, as well as with rho, to activate Egfr signaling (Guichard, 2000).

The data presented thus far suggest that the Brho protein can function early during oogenesis by activating Egfr signaling in follicle cells adjacent to the oocyte where brho is expressed. As a possible mechanism, it is proposed that Brho might promote processing or activation of the Grk protein in the oocyte to stimulate Egfr expressed in adjacent follicle cells. Consistent with the idea that mGrk, like mSpi, requires activation, the mGrk protein does not exhibit any activity when misexpressed in the wing. In contrast, an artificially truncated version of Grk, GrkDTM, can activate Egfr both in the wing and in follicle cells. In order to determine whether activation of mGrk involves Star, as has been observed for mSpi, Star and gurken were coexpressed during wing development (Guichard, 2000).

Coexpression of mgrk and Star results in a strong ectopic vein phenotype, which is greater than that caused by coexpression of mspi and Star. This finding supports the view that the Grk EGF ligand can be activated through a mechanism similar to that of mSpi (Guichard, 2000).

To determine whether Brho can also participate in activating Grk, UAS-brho and UAS-mgrk were co-expressed in the wing. The ectopic vein phenotype resulting from the coexpression of brho and mgrk is significantly stronger than that caused by brho alone, indicating that Brho can activate the mGrk precursor. A synergistic effect between brho and mspi was observed, similar to that which has been observed between rho and mspi . The phenotypes resulting from coexpressing brho + mgrk are significantly stronger than those from coexpressing brho + mspi; however, it is not believed that this necessarily reflects a preference of Brho for activating Grk versus Spi, since coexpression of UAS-Star with these ligands also results in a much stronger phenotype with Grk than Spi. These data suggest rather that the UAS-mGrk construct may be expressed more efficiently or at higher levels than the UAS-mspi construct. Also, it was not possible in these experiments to determine whether there was a significant increase in the severity of the phenotype resulting from coexpression of UAS-rho with either UAS-mspi or UAS-mgrk in the wing since UAS-rho generates a very strong ectopic vein phenotype when misexpressed alone (Guichard, 2000).

It is concluded that brho functions like rho by collaborating with Star in activating Egfr/MAPK signaling. Brho can potentiate the activity of both mSpi and mGrk EGF ligands, consistent with the possibility that Brho may activate Grk to promote Egfr/MAPK signaling and define posterior fates in the early follicular epithelium (Guichard, 2000).

DEVELOPMENTAL BIOLOGY

Star: Biological Overview | Regulation | Effects of Mutation | References

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