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, mbGrkDelta19AAmyc, 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 Star is necessary for trafficking/secretion of the ligand. However, Star is not able to cleave Grk in the 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).
Rho-1/Brho may facilitate Grk proteolysis either by activating or recruiting a yet unknown protease. By analogy to the processing of mammalian Egfr ligands, Grk cleavage may be catalyzed by an ADAM-like metalloprotease. Although these molecules are present in Drosophila, nothing is known yet about their functions. An alternative hypothesis, is that despite the absence of known protease domains in their sequences, Rho-1 and Brho themselves may have proteolytic activity. The subcellular localization of Brho, as observed for mature TACE (ADAM17), is predominantly in intracellular compartments. In addition, and directly relevant to this hypothesis, Presenilins, which define another subfamily of seven-pass transmembrane proteins, have been proposed to encode proteases. In Drosophila, Presenilin may be directly responsible for the proteolysis of the intra-transmembrane domain of Notch (Ghiglione, 2002 and references therein).
One of the striking features of Rho-related proteins is that amino acid sequence conservation is most prominent in the predicted TM regions that contain some invariant charged residues. This suggests the presence of a hydrophilic pocket that might constitute an enzymatic active site or a channel, as observed in Presenilins. This model is further supported by the recent finding that the TM domain of Spi is important for its functional interaction with Rho-1 (Ghiglione, 2002 and references therein).
rho-related genes have been found in organisms from diverse kingdoms including C. elegans, rat, human, Arabidopsis, sugar cane, yeast and bacteria. The data suggest that Brho, like Rho-1, promotes Egfr signaling by activating TGFalpha-like ligands. Since RTKs have not been found in plants, yeast or bacteria, the rho-related genes in these organisms presumably serve other functions. It will be interesting to determine whether the activities of these Rho-related proteins are similar to those of Rho-1 and Brho, such as promoting the processing of proteins (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).
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).
The ability of Rhomboids 1-4 to catalyse Spitz cleavage in the tissue culture assay suggested that all may be involved in activating the EGF receptor in vivo. This has been clearly demonstrated for Rhomboid-1, was genetically determined in the case of Rhomboid-3, and has been proposed for Rhomboid-2. To investigate this further, the potential activity of Rhomboids 2-4 in vivo was compared by overexpressing them in developing Drosophila tissues. In all cases examined, Rhomboids 2-4 cause similar phenotypes to Rhomboid-1, consistent only with EGF receptor hyperactivation. When expressed in the developing wing, for example, all core Rhomboids produce ectopic and thickened vein phenotypes similar to those observed for Rhomboid-1. This phenotype was modified predictably by mutations in other members of the EGF receptor pathway. Furthermore, as in cell culture assays, all four Rhomboids are synergistic with the co-expression of Star. In all cases, UAS Rhomboids 1 and 3 produce consistently strong wing phenotypes, whereas Rhomboids 2 and 4 are weaker. Similar results were obtained in the eye, follicle cells of the ovary and the embryo. Importantly, no other phenotypes were observed in eyes, wings or embryos expressing Rhomboids, suggesting that they do not affect any other pathways. If, for example, the previously uncharacterized Rhomboids 2 or 4 caused the activation of other signaling pathways, their ectopic expression would lead to additional phenotypes. These observations confirm that Rhomboids 2-4 contain the same proteolytic activity as Rhomboid-1; furthermore, the absence of phenotypes associated with other pathways strongly suggests that Rhomboids 1-4 are all dedicated to regulating EGF receptor signaling. These data demonstrate that Rhomboids 1-4 all share proteolytic activity against the ligand Spitz (Urban, 2002).
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