iRhoms Lack Proteolytic Activity
Biochemical and structural analysis of rhomboid proteases has identified the two essential catalytic residues as being serine-201 and histidine-254 (numbered according to E. coli
GlpG) (Wang et al., 2006
). Phylogenetic alignment of eukaryotic rhomboid-like proteins identified a well-conserved subfamily in which one or both of these catalytic residues were missing, despite otherwise having clear sequence and topological similarity to the active rhomboids (A) (Lemberg and Freeman, 2007
). The human genes have been named Rhbdf1 and Rhbdf2, but the generic term iRhoms has been proposed to distinguish them from the active proteases (Lemberg and Freeman, 2007
). The degree of conservation of these iRhoms, which are only found in metazoans, suggests that they are under evolutionary selective pressure, but as they are not predicted to be active proteases, their function is unclear. iRhoms have three prominent distinctions from active rhomboids: a much longer N-terminal cytoplasmic domain, a large and extremely conserved loop domain (the iRhom homology domain) between the first two transmembrane domains, and an invariant proline residue that is N terminal to the expected location of the catalytic serine (i.e., GPx replacing the GxS rhomboid catalytic motif). The significance of the iRhom-specific proline was investigated by generating variants of active rhomboids in which the residue immediately before the catalytic serine was mutated to proline. This mutation abolished the enzymatic activity of Drosophila
Rhomboid-1 and strongly reduced mouse RHBDL-2 activity in a cell culture assay (B–1D). The proline mutation also virtually abolished the in vitro activity of the purified bacterial rhomboid AarA (Strisovsky et al., 2009
), demonstrating that it directly disrupts the enzyme (E).
iRhoms Are Inactive Members of the Rhomboid-like Family
This prediction that iRhoms are themselves catalytically inert was tested by analyzing Drosophila
iRhom, human iRhom1 (Rhbdf1), and mouse iRhom2 (Rhbdf2) in a cell culture proteolysis assay. No proteolytic activity was detected against the Drosophila
EGFR ligands Gurken or Spitz, nor against mouse EGF (B–1D), all substrates of multiple rhomboids (Urban et al., 2002a, 2002b; Adrain et al., 2011
). We have further tested Drosophila
and mammalian iRhoms against a variety of other rhomboid substrates without detecting any proteolytic activity (data not shown). Overall, these data support the idea that iRhoms have no proteolytic activity and demonstrate that their characteristic proline is sufficient to disrupt the rhomboid active site.
Drosophila iRhom Is Expressed in Neuronal Cells and Is Located in the ER
Unlike most sequenced metazoans, Drosophila
has only a single iRhom, encoded by the rhomboid-5
gene. In situ hybridization showed that rhomboid-5/iRhom
RNA is expressed exclusively in the central nervous system of embryos: transcripts are strongly detected in the ventral nerve cord and brain (A). In the third instar larva, rhomboid-5/iRhom
expression was also largely restricted to neural tissue: specifically behind the morphogenetic furrow in the developing eye (B), where the adult retina develops, and in the embryonic brain, with elevated levels in the optic lobes (C). In adults, too, iRhom RNA is highly enriched in the nervous system and brain (http://www.flyatlas.org
Expression and Cellular Location of iRhoms
Individual active rhomboid proteases have specific locations in the Golgi apparatus and plasma membrane (D) or in mitochondria (Lee et al., 2001; Urban et al., 2002a; McQuibban et al., 2003
). We used COS7 cells to determine the localization of HA-tagged Drosophila
iRhom, human iRhom1, and mouse iRhom2. All were located specifically in the endoplasmic reticulum (ER), as demonstrated by characteristic perinuclear and reticular staining and by colocalization with the ER marker protein disulphide isomerase (PDI) (D). This is supported by an earlier observation that human iRhom1 is located in the ER in HNSCC 1483 cancer cells (Zou et al., 2009
Drosophila iRhom Mutants Phenocopy Neuronal EGFR Activation
To investigate iRhom function, we used homologous recombination to generate a null mutant of Drosophila iRhom
( available online). None of the three mutant lines that we isolated had discernible developmental defects. Homozygous mutations did, however, cause a severe decrease in the daytime activity pattern of adult flies compared to wild-type or heterozygous controls (A and Movie S1
). The specificity of this phenotype was confirmed by transheterozygotes between the targeted mutation and a pre-existing deletion for the region having the same phenotype (A). Furthermore, UAS-iRhom
expressed under the control of the neuron-specific ELAV-Gal4
driver fully rescued the activity of the mutant flies (B), whereas expression in muscle cells, under the control of how24B-Gal4
, did not. This confirms the specificity of the phenotype and demonstrates that iRhom is required specifically in neurons, a result that is consistent with the nervous system-restricted expression pattern (A–2C).
Generation of iRhom/rhomboid-5 Mutant Drosophila, Related to
Loss of iRhom Results in Increased Sleep
During the day, iRhom
mutant flies sometimes do not move for periods of 1 hr, which is highly unusual. Prolonged periods of inactivity could, in principle, be caused by increased resting (a sleep-like state) or an impaired ability to move. To distinguish these two possibilities, we measured the activity of flies during periods of wakefulness. During their relatively few active periods (147 over the course of the experiment, compared to 820 in the control), mutants showed no reduction of movement; in fact, in those periods, they moved slightly more than wild-type or heterozygous controls (C). To confirm this, we analyzed video recordings and found mutants and wild-type to have the same walking speed (D). This indicates that iRhom
mutant flies can move normally and implies that their overall inactivity is due to an increase in periods of inactivity between active episodes. Consistent with this, sleep, typically defined as periods of inactivity greater than 5 min (Greenspan et al., 2001
), was significantly increased in iRhom
This same inactivity phenotype is caused by activation of EGFR signaling upon transient CNS expression of Rhomboid-1 and Star (Foltenyi et al., 2007
). This is significant because of the well-established functional relationship between active rhomboids and EGFR activation (Wasserman et al., 2000; Urban et al., 2001
). Our results are therefore consistent with loss of Drosophila
iRhom in the nervous system leading to activation of EGFR signaling, thereby decreasing daytime activity. This would imply that, under wild-type conditions, iRhom promotes wakefulness by inhibiting EGFR activity.
Drosophila iRhom Is a Specific Inhibitor of EGFR Signaling
The cellular mechanism by which EGFR signaling in the CNS regulates Drosophila sleep patterns is not yet understood, so to investigate further the genetic relationship between iRhoms and EGFR, we used the well-characterized systems of imaginal disc development. We looked for genetic interactions to test the hypothesis that iRhoms act as physiological inhibitors of EGFR activity. Halving the dose of iRhom, which alone caused no phenotype, strongly enhanced the rough eye caused by EGFR hyperactivity induced by Rhomboid-1 overexpression (A). Conversely, removal of iRhom suppressed the eye phenotype caused by reduction of EGFR signaling, for example when Argos, Sprouty, or a dominant-negative form of the EGFR was overexpressed in the eye (B). Both of these synergistic interactions imply that iRhom inhibits EGFR signaling; this was directly demonstrated by iRhom overexpression (using GMR-Gal4) suppressing the rough eye caused by the overexpression of active Rhomboid-1 (C).
Drosophila iRhom Inhibits EGFR Signaling
In another set of experiments, we examined the consequences of iRhom heterozygosity in combination with reduction in the known EGFR inhibitory molecules Sprouty and Argos. Removing one copy of sprouty triggered a mild extra wing vein phenotype in 6.4% of adult flies grown at 29°C; this was enhanced to 74.5% when the dose of argos was simultaneously halved (D). Similarly, halving iRhom strongly enhanced the sprouty heterozygous wing phenotype: 62.5% of wings had mild, and 3.65% had strong extra vein phenotypes (D). The additive effect of all three genes was further demonstrated in the triple-heterozygote combination, wherein mild (+) and severe (++) extra vein phenotypes were further increased to 87.5% and 10.3%, respectively (D). Together, all of these synergistic genetic interactions indicate that iRhom acts in the same pathway as Argos and Sprouty and is an inhibitor of EGFR signaling.
Importantly, we also tested whether other developmentally significant signaling pathways were similarly perturbed by changes in the dose of iRhom. In an extensive series of experiments, we found no evidence for genetic interactions with Wg, Notch, Hedgehog, or Dpp signaling (Table S1
). Overall, we conclude that Drosophila
iRhom inhibits EGFR activity in vivo; that this role is particularly prominent in the nervous system but can be detected in the developing wing and eye under sensitized conditions; and that, although we cannot rule out a role in pathways that have not been tested, this effect is largely specific to the EGFR-signaling pathway.
To confirm that disruption of the EGFR pathway also can explain the sleep-like iRhom phenotype, we looked for similar genetic interactions affecting activity (E). Reduction of EGFR signaling by RNAi knockdown of the EGF receptor itself, or its activating ligand Spitz, significantly rescued the iRhom loss-of-function phenotype, supporting the idea that the lethargy in the mutant flies is indeed caused by excess EGFR activity (E).
Drosophila and Mammalian iRhoms Specifically Inhibit Rhomboid-Triggered Ligand Release
The genetic data described above demonstrate a physiological role for Drosophila
iRhom but cannot provide direct mechanistic insight. For this, we turned to cell culture, asking whether iRhoms can interfere with the function of active rhomboid proteases. When Gurken, a Drosophila
EGF family ligand, and Drosophila
Rhomboid-1 are coexpressed in COS7 cells, Gurken is cleaved and its extracellular domain is released into the culture medium (A) (unlike Spitz, Gurken does not require the action of Star [Yogev et al., 2008
]). Coexpression of Drosophila
iRhom inhibited the release of soluble Gurken induced by Rhomboid-1 (A). This effect was specific, to the extent that the release into the medium of two other proteins, Delta, a transmembrane protein subject to metalloprotease shedding, and Wnt3A, a secreted protein, was unaffected by iRhom expression (B and A). We also ruled out a different kind of nonspecific effect by coexpressing with Gurken and Rhomboid-1 similar levels of an unrelated polytopic ER protein, Unc93B (Brinkmann et al., 2007
). This caused no reduction of Gurken release (C), implying that the iRhom effect was not a nonspecific consequence of overexpressing a polytopic membrane protein (for example, caused by overload of the ER). We also found that the release of Spitz, another Drosophila
EGFR ligand, is similarly blocked by iRhom coexpression (data not shown). Together, these results show that Drosophila
iRhom specifically inhibits EGFR ligand release from cells. Because iRhoms are conserved in all metazoans, we asked whether their function might be conserved between Drosophila
and mammals. When human iRhom1 (data not shown) or mouse iRhom2 were coexpressed with mouse EGF and the mouse active rhomboid, RHBDL2 (Adrain et al., 2011
), secretion of soluble EGF was inhibited (D), implying that both mammalian iRhoms had a similar function to their Drosophila
iRhom Inhibits Secretion of EGF Family Ligands
iRhom Did Not Have an Impact on WNT3a Secretion Intracellular WNT3a or Delta Levels, Related to
iRhoms Specifically Reduce Intracellular Levels of EGF Family Ligands
The specific inhibitory effect of iRhom on the accumulation of soluble Gurken in the cell culture medium was also reflected in cell extracts, but in this case, we detected two distinct effects. First, iRhom coexpression reduced Gurken cleavage by Rhomboid-1, expressed as relative substrate conversion (E). Second, the total level of intracellular Gurken, intact and cleaved, was reduced by iRhom coexpression (E). Again, controls imply that this was a specific effect: the polytopic ER protein Unc93B did not reduce Gurken cleavage or intracellular levels (F), and neither Wnt3A nor Delta were destabilized by iRhom expression (B and S2C). Finally, all of these effects were efficiently rescued by the additional expression of an ER-targeted version of Rhomboid-1, but only slightly rescued by high levels of wild-type Rhomboid-1, which is located in the Golgi apparatus (G). This result is important for a number of reasons. First, it further demonstrates that the iRhom effect is not caused by a nonspecific disruption of the secretory pathway. Second, once Gurken has been solubilized by an active rhomboid, it is no longer subject to iRhom-mediated destabilization. Third, the fact that ER-restricted Rhomboid-1 rescues much more efficiently than Golgi-localized Rhomboid-1 argues that iRhom function occurs in the ER, not later in the secretory pathway. The slight rescue by wild-type Rhomboid-1 presumably reflects the low steady-state level in the ER as it is trafficked to the Golgi apparatus.
iRhom Acts in Absence of Active Rhomboid
Two classes of mechanism could explain our data: the iRhoms might either inhibit the function of active rhomboid proteases, for example by a dominant-negative effect, or could act directly on substrates, destabilizing them and preventing them from being cleaved by proteases. We took advantage of the absence of endogenous rhomboid activity in COS7 cells (Urban and Freeman, 2003; Adrain et al., 2011
) to distinguish these models by testing the effect of iRhom in the absence of an active rhomboid. Human iRhom1 or mouse iRhom2 were coexpressed with EGF and other EGF family ligands. Under these conditions, ligand release was dependent on ADAM family metalloproteases, which were not chemically inhibited (as they were in some previous experiments). Strikingly, the secretion and intracellular levels of all EGFR ligands tested (EGF, TGFα, Epiregulin [EPR], Amphiregulin [AREG], Betacellulin [BTC], and Neuregulin 4 [Nrg4]) were downregulated by the expression of either iRhom (A and 6B and A–S3D). Two control proteins, prolactin, a constitutively secreted protein, and Delta, a membrane protein that is subject to metalloprotease shedding, but not cleaved by rhomboids, were unaffected by iRhom expression (C and E). As before, specificity was demonstrated by showing that Unc93B did not cause the same effect (A–6C and A–S3D). By repeating the experiment in HeLa cells, we also confirmed that the specific downregulation of EGF by iRhoms was not limited to COS cells (F). These experiments clearly demonstrate that iRhoms can downregulate mammalian EGF family ligands (not all of which are rhomboid substrates [Adrain et al., 2011
]) in the absence of any active rhomboid, thereby strongly supporting a model whereby iRhoms act directly on EGFR ligands (or possibly other clients), rather than by inhibiting the rhomboid enzymes themselves.
iRhoms Induce Degradation of EGFR Ligands Independently of Active Rhomboids
iRhoms Reduce Secretion and Intracellular Levels of Multiple EFGR Ligands, Related to
We further tested this model by examining whether a mutant version of EGF (A1031F) that cannot be cleaved by rhomboids but is susceptible to ADAM release (Adrain et al., 2011
) was sensitive to iRhom coexpression. Both human iRhom1 and mouse iRhom2 downregulated EGFA1031F
and inhibited its release into the medium of COS7 cells (D). This further confirms that iRhoms can act on proteins that are not themselves substrates of rhomboid proteases.
Is the iRhom effect simply a consequence of an interaction between a catalytically dead rhomboid and a potential substrate? We tested this by investigating whether catalytic mutants of mouse RHBDL2 would mimic the iRhom effect on EGF. No downregulation of EGF was detected upon expression of an ER-targeted mutant of RHBDL2 (E).
In summary, these experiments lead us to conclude that Drosophila and mammalian iRhoms share a conserved function; that iRhoms inhibit rhomboid-catalyzed release of growth factors by acting on the potential rhomboid substrates, rather than by inhibiting the enzymes themselves; and that the iRhoms have a specific function beyond just being catalytically inactive rhomboid proteases. An important further conclusion is that, although iRhoms show significant specificity, they can act on proteins that are not direct rhomboid substrates.
iRhoms Trigger Proteasomal Degradation of Rhomboid Substrates
The observed reduction of EGF family ligands induced by iRhoms suggested that they might be degraded in the cell. We therefore asked whether inhibition of the proteasome would suppress iRhom-induced downregulation. Myc-tagged mouse EGF was coexpressed with iRhom1 in the presence or absence of proteasomal inhibitors. As expected, iRhom expression caused a substantial reduction of EGF, both in cell extracts and secreted into the culture medium. This reduction was completely rescued by treatment with MG132, implying that the destabilization of EGF depended on proteasomal activity (A). This result was confirmed with another proteasome inhibitor, lactacystin (A). We also used qPCR to show that the observed increases in EGF levels were not caused by nonspecific transcriptional effects of the proteasome inhibitors (B and S4C). Because EGF resides in the secretory pathway, it is not directly accessible to proteasomes in the cytoplasm. However, proteins can be extracted from the ER for proteasomal destruction in a process called ER-associated degradation (ERAD) (Brodsky and Wojcikiewicz, 2009
). The location of iRhoms in the ER is consistent with a potential role in promoting ERAD. Interestingly, even in the absence of iRhom, MG132 treatment also slightly increased the steady-state level of intracellular EGF (A), implying that, even under normal conditions, some EGF is degraded by the proteasome.
iRhoms Destabilize EGFR Ligands by ERAD
iRhoms Destabilize EGFR Ligands by ERAD, Related to
The conclusion that iRhom drives EGF into the ERAD machinery suggests that the two proteins might directly interact in the ER. Indeed, Flag-tagged EGF was specifically coimmunoprecipitated by HA-tagged human iRhom1 and mouse iRhom2, whereas control proteins, TGN36 and prolactin, were not (B).
To monitor the kinetics of intracellular EGF, which can leave the cell by secretion or be degraded by the proteasome, we performed a pulse-chase experiment (C). Because iRhom is expressed before the label is added, the iRhom effect is already apparent at the earliest stage of the chase, complicating the interpretation of this experiment. Nevertheless, this kinetic analysis is fully consistent with the steady-state data (A and A). In untreated cells, a pulse of labeled EGF (C, blue diamonds) disappears from cells during a 3 hr time course. As expected, coexpression of iRhom1 reduces the level of EGF (C, green triangles); again, all EGF disappears during the 3 hr chase. Addition of MG132 to inhibit the proteasome increases the initial level of EGF, confirming that ERAD contributes to EGF homeostasis (A), but most of the EGF is still secreted by the cell over 3 hr (C, red squares). Interestingly, coexpression of iRhom in cells in which the proteasome is inhibited (C, purple crosses) slows down EGF secretion. This partial ER-anchoring effect by iRhom when it cannot promote ERAD is consistent with its direct binding to EGF (B).
Finally, the in vivo relevance of this model was tested by using Drosophila genetics to look for evidence that ERAD contributes to the normal regulation of EGFR signaling. Inhibition of EGFR signaling by overexpression of either Sprouty or Argos was suppressed by RNAi knockdown of the ERAD factors Hrd1 (D) or EDEM2 (D). This implies that ERAD does indeed regulate EGFR signaling in the Drosophila eye. Overall, these data suggest that EGF levels in Drosophila and perhaps in mammals are normally kept in balance by low-level ERAD and that iRhoms exploit this mechanism to regulate growth factor signaling (E).