Gain of Function EGFR-Ligand Shedding Experiments with ADAM 8, -9, -10, -12, -15, -17, and -19
Previous loss of function experiments in mouse embryonic fibroblasts identified ADAM 10 and -17 as the main EGFR-ligand sheddases. Because ADAMs are known to be dysregulated in diseases such as cancer, we performed gain of function experiments with ADAM 8, -9, -10, -12, -15, -17, or -19 to determine which of these proteases can cleave which EGFR-ligands when overexpressed. To facilitate detecting EGFR-ligand sheddase activity for these ADAMs, all experiments were performed in cells lacking the major endogenous sheddase for each ligand (ADAM17 for TGFα, HB-EGF, AR, and EPR; ADAM10 for EGF and BTC; Sahin et al., 2004
). Constitutive and PMA-stimulated shedding of TGFα and EPR from Adam17
−/−-deficient cells could be rescued by ADAM17, but not ADAM 8, -9, -10, -12, or -15 (A). Shedding of HB-EGF and AR was increased by overexpressing ADAM17 and ADAM8, but not the other ADAMs tested here (, C and D). ADAM17-dependent shedding of HB-EGF and AR was consistently enhanced by PMA, albeit not to the same degree as in wild-type control mEF cells or COS-7 cells, whereas ADAM8-dependent release of these ligands was not. Processing of EGF in Adam10
−/− cells was increased by ADAM 8, -9, -10, -12, -17, and -19, but not by ADAM15 (E, expression of mature ADAM15 was confirmed by Western blot analysis; data not shown). EGF shedding was not significantly stimulated by PMA in any of the rescue experiments, including those with ADAM17. Finally, ADAM 8, -10, -12, -17, and -19 could also shed BTC, whereas overexpressed ADAM 9 or -15 did not, although the expression of mature forms of both ADAMs was confirmed by Western blot analysis (data not shown). In all cases where increased shedding was observed, it did not require stimulation. Thus, these experiments demonstrate which ADAMs can cleave which EGFR-ligands constitutively, which may be particularly relevant for diseases where ADAM expression is up-regulated (please note, however, that these experiments do not allow a quantitative comparison of how efficiently different ADAMs shed various EGFR-ligands).
Figure 1. Evidence for a contribution of multiple ADAMs to EGFR-ligand shedding. AP-tagged EGFR-ligands (A, TGFα; B, EPR; C, HB-EGF; D, AR; E, EGF; and F, BTC) were cotransfected either with empty vector (−) or with an expression vector for ADAM8 (more ...)
PMA-dependent Stimulation of EGFR-Ligand Shedding
Among the widely expressed and catalytically active ADAMs tested here, ADAM17 emerged as the only PMA-responsive sheddase, at least with respect to release of TGFα, HB-EGF, AR, and EPR. We therefore focused on ADAM17 and two of its substrates, TGFα and AR, for an evaluation of how this phorbol ester regulates ectodomain shedding. Two recent studies reported that PMA enhances maturation and prodomain processing of ADAM17 within 10 min of PMA stimulation (Nagano et al., 2004
; Soond et al., 2005
). However, PMA stimulation did not detectably increase the levels of HA-tagged ADAM17 (A, top left) or endogenous ADAM17 (A, top right, pro-ADAM17, open arrowhead; mature ADAM17, black arrowhead) that could be labeled with a membrane-impermeable biotinylation reagent under the conditions used here, even though it strongly stimulated ADAM17-dependent ectodomain shedding in COS-7 cells (see below; D). Moreover, PMA stimulation did not increase the maturation of HA-tagged ADAM17 or endogenous ADAM17 (A, bottom). When experiments identical to those shown in A were performed in simian virus 40 (SV40) transformed wild-type mouse embryonic fibroblasts, we also did not detect an increase in the maturation of endogenous or transfected HA-tagged ADAM17 after stimulation with PMA (see Supplemental Figure 1). In a pulse-chase experiment in COS-7 cells, the processing of wild-type HA-tagged ADAM17 as well as a Myc-tagged mutant ADAM17 lacking its cytoplasmic domain (A17b) was also not increased after PMA stimulation for 2 h (B).
Figure 2. Effect of PMA on the maturation of ADAM17 in COS-7 cells. (A) COS-7 cells transfected with HA-tagged ADAM17 (A17HA, left) or not transfected (right) were incubated with 500 ng/ml PMA for 10 and 30 min followed by labeling of molecules on the cell surface (more ...)
Figure 3. Subcellular localization of EGFR-ligands and effect of PMA stimulation on shedding of TGFα and AR (A) AP-tagged TGFα and ADAM17-cotransfected in wild-type mEF cells were incubated in the absence (a–c) or presence of PMA (25 ng/ml, (more ...)
Because PMA did not have a detectable effect on ADAM17 maturation in COS-7 cells in this study, we asked whether it might instead act on EGFR-ligands. An immunofluorescence analysis showed that TGFα and ADAM17 colocalize in unstimulated cells in a perinuclear compartment (A, a–c). The perinuclear staining of TGFα, overlaps with that of wheat germ agglutinin, a marker for the trans-Golgi network (TGN; data not shown). In addition, diffuse staining of TGFα in vesicles throughout the cell, presumably corresponding to endoplasmic reticulum (ER) staining, can be detected in longer exposures (data not shown). PMA stimulation did not affect the localization of ADAM17 or TGFα (A, d–f). Interestingly, among the other EGFR-ligands whose shedding by ADAM17 can be stimulated by PMA, only EPR had a very similar localization as TGFα (B, d–f). In contrast, HB-EGF and AR had a more prominent cell surface localization, although substantial amounts of these growth factors also colocalized with TGFα (B, g–l). EGF and BTC, whose shedding is not stimulated by PMA, were mainly found on the cell surface, although there was some colocalization with TGFα in intracellular compartments (B, m–r). Because all ligands were detected via their alkaline phosphatase tag, we confirmed that two differently tagged forms of TGFα (AP and HA-tagged) colocalized intracellularly (B, a–c), which corroborates that the AP-tag does not change the localization of this EGFR-ligand. Because immunofluorescence analysis showed that substrates of ADAM17 (TGFα, HB-EGF, EPR, and AR) accumulate either in a perinuclear compartment or on the cell surface, the predominant subcellular localization of EGFR-ligands is presumably not a main determinant of the response of ADAM17 to PMA stimulation.
Another possible mechanism underlying PMA stimulation is increased transport of EGFR-ligands through the secretory pathway. The AP-tag contains N-linked carbohydrate residues; therefore, acquisition of resistance to EndoH indicates that AP-tagged EGFR-ligands have passed through the medial-Golgi apparatus. In AP-tagged TGFα, its major proform at 75 kDa (C, a, black arrow) was completely EndoH sensitive, whereas only the small amount of a slower migrating form of pro-TGF at ~79 kDa was resistant to EndoH (C, a, open arrow). Both forms could be deglycosylated by PNGase F (C, a, asterisk), which removes all N-linked carbohydrate residues. Similar experiments with AR showed substantial concentrations of EndoH-resistant pro-AR (C, b, open arrow) as well as small amounts of EndoH-sensitive pro-AR (C, b, asterisk). Thus, more pro-AR than pro-TGFα accumulates after passage through the medial-Golgi apparatus, which is consistent with the immunofluorescence studies described above. PMA stimulation only removed the small amount of EndoH-resistant 79-kDa pro-TGFα (D, a, lanes 1 and 2, white arrow), whereas the 75-kDa EndoH-sensitive form of TGFα seemed unaffected (D, a, lane 3, white arrow). Processing of the 79-kDa pro-TGFα could be prevented by the hydroxamate metalloprotease inhibitor BB94 in PMA-stimulated cells (D, a, lane 4). In contrast to TGFα, a majority of pro-AR was removed after PMA stimulation, and this could also be blocked by BB94 (D, b). Evidently, PMA only or mainly triggers shedding of EndoH-resistant EGFR-ligands that have already passed the medial Golgi apparatus, and has little or no effect on the intracellular maturation of these ligands.
To more directly address the role of PMA in increasing the intracellular maturation and transport of ADAM17 or its substrates to the trans
-Golgi network, we tested whether BFA, a fungal toxin that blocks the secretory pathway by collapsing the Golgi apparatus into the ER (Lippincott-Schwartz et al., 1989
), blocks PMA stimulated shedding of these EGFR-ligands. Remarkably, BFA treatment for 1 h had little effect on the PMA-stimulated shedding of AR (8% decrease) or TGFα (28% decrease), and it did not detectably affect their constitutive release (E, a and b). Under identical conditions, BFA strongly reduced the release of soluble alkaline phosphatase (AP) (84%; F). PMA-stimulated shedding of TGFα and AR decreased substantially in the second hour in the absence of BFA, and it returned to constitutive levels after 3 h (E). BFA had a more pronounced effect on stimulated shedding after 3–4 h, suggesting that it prevents replenishment of pro-TGFα and pro-AR once the EndoH-resistant pool of these molecules is consumed. Secretion of soluble AP was not stimulated by PMA (data not shown), providing additional evidence against a general effect of PMA on the constitutive secretory pathway under the conditions used here. Thus, short-term PMA stimulation affects only or mainly ADAM17 as well as substrates that have already passed the medial
-Golgi apparatus, without a requirement for increased intracellular maturation. Finally, when we analyzed the concentration of endogenous ADAM17 by Western blot in wild-type SV40-transformed mEF cells treated with 25 ng/ml PMA, in the presence or absence of BFA, we found similar amounts of pro-ADAM17 and mature ADAM17 in cells treated with PMA or PMA/BFA for up to 2 h as in untreated cells (data not shown). Thus, even though a higher concentration of PMA (100 ng/ml) stimulated down-regulation of mature ADAM17 (Doedens and Black, 2000
), the lower concentration of PMA used in this experiment (25 ng/ml) did not.
Chimera between ADAM10 and -17 Address the Role of Different ADAM Domains in PMA-stimulated Shedding of TGFα
Because ADAM17 responds strongly to 1-h PMA stimulation, whereas ADAM10 does not, we used chimera between mouse ADAM17 and ADAM10 to determine which domains of ADAM17 are essential for PMA-stimulated shedding of TGFα via rescue experiments in Adam17−/− cells (see A for a diagram of the chimera, and a key to the nomenclature, T, TNF-α converting enzyme/ADAM17; K, kuzbanian/ADAM10). Constitutive and PMA-stimulated TGFα shedding was restored by wild-type ADAM17 as well as mutants in which the transmembrane domain and cytoplasmic domains of ADAM17 had been replaced by the corresponding domains of ADAM10 (TTKK), and to a lesser extent (~50%) by ADAM17 lacking its cytoplasmic domain (A17b, TTT; B). None of the other chimera in which only the metalloprotease or disintegrin domains had been swapped (KTTT, TKKK, and TKTT), were able to rescue PMA-stimulated TGFα shedding. The protein expression levels resulting from transfections of COS-7 cells with 1 μg each of plasmids coding for wild-type ADAM10 or ADAM17 or either of the four mutants was determined by Western blot analysis. As shown in Supplemental Figure 2A, wild-type ADAM17 and TKTT had the highest expression, ADAM10, TKKK, and TTKK were expressed at intermediate levels, whereas the expression of KTTT was low but still clearly detectable (please see B for a side-by-side comparison of the expression of wild-type ADAM17 and ADAM17b). In addition, immunofluorescence analysis showed a comparable staining pattern of the four mutants (see Supplemental Figure 2B). Thus, PMA stimulation requires an intact ectodomain of ADAM17 but not its cytoplasmic or transmembrane domains.
Figure 4. Rescue of constitutive and PMA-stimulated shedding of TGFα in Adam17−/− cells by chimera between ADAM10 and 17 (A) Diagram of chimeras between ADAM10 and ADAM17 (K, KUZ or kuzbanian, ADAM10; T, TACE, TNFα-converting enzyme, (more ...)
Chimera between TGFα and EGF or BTC Define the Requirements for Stimulated Shedding by ADAM17
We next focused on chimera between TGFα and EGF or BTC to determine which domains of TGFα are required for stimulated shedding by ADAM17 (a diagram of the chimera and a key to the nomenclature is shown in A). Each chimera was cotransfected into Adam17−/− cells with empty vector or with ADAM17. Overexpressed ADAM17 only slightly enhanced processing of EGF (B; also see ) but could release relatively high amounts of TGFα from Adam17−/− cells. Moreover, ADAM17-dependent shedding of TGFα was strongly stimulated by PMA (B; also see ). Shedding of the chimera with the EGF ectodomain and TGFα transmembrane and cytoplasmic domain (ETT) was similar to that of TGFα, although there was a slight increase in constitutive shedding in the absence of ADAM17. Shedding of EET was inefficient and was only slightly increased upon rescue with wild-type ADAM17, whereas very little ADAM17-dependent shedding of TEE was observed. However, placing the ectodomain and juxtamembrane domain of TGFα on the cytoplasmic domain of EGF (TTE) restored ADAM17-dependent constitutive and PMA-induced shedding, although not to the same level as seen with wild-type TGFα. Moreover, ADAM17 induced slightly more shedding of TET compared with TEE. Finally, constitutive and stimulated ADAM17-dependent shedding of ETE was similar to that of TTE, demonstrating the cleavage site of TGFα is necessary and sufficient for constitutive and PMA-dependent processing by ADAM17. Shedding experiments with chimera between BTC and TGFα yielded largely comparable results (C), with the exception that ADAM17 had little or no effect on shedding of two chimera in which the ectodomain of BTC was positioned next to the cleavage site of TGFα (BTT, BTB), suggesting that the EGF-like domain in BTC blocks access to the TGFα cleavage site. The expression of all chimeras was confirmed by immunofluorescence analysis (see Supplemental Figure 3).
Figure 5. Chimera between TGFα and EGF or BTC define the requirements for selective PMA-dependent and constitutive shedding of TGFα by ADAM17. (A) Diagram of chimeras between TGFα and EGF or BTC. Domain components of EGF (or BTC) and TGFα (more ...)
Calmodulin Inhibitor- and Calcium Ionophore-stimulated Shedding of EGF and BTC by ADAM10
Because ADAM17 lacking its cytoplasmic domain could still be stimulated by PMA (also see Reddy et al., 2000
), arguing that a direct interaction with cytoplasmic molecules is not necessary for this effect, we tested whether ADAM 10 or -17 require their cytoplasmic domain to respond to a different stimulus, the calcium ionophore IM, and the calmodulin inhibitor trifluoroperazine (TFP). Both have previously been shown to activate ADAM10 (Nagano et al., 2004
; Sanderson et al., 2005
). We found that shedding of EGF and BTC in Adam10
−/− cells rescued with wild-type ADAM10 was enhanced by TFP (A). Little effect of TFP on EGF or BTC shedding was seen in Adam10
−/− cells transfected with empty vector or the inactive ADAM10 E > A mutant, corroborating that TFP-stimulated shedding of EGF and BTC depends mainly on ADAM10 (A).
A previous study suggested that IM stimulates shedding of CD44 by enhancing the intracellular maturation of ADAM10 (Nagano et al., 2004
). However, stimulation with IM for 10 or 30 min did not affect the maturation of HA-tagged ADAM10 or endogenous ADAM10 that could be cell surface biotinylated or detected by Western blot (B). Furthermore, in an immunofluorescence analysis, the subcellular localization of ADAM10 and BTC was indistinguishable in IM-treated mEF cells compared with untreated controls (C, a–f), and both ADAM10 and ADAM17 colocalize in these cells (E, g–i).
The ability of ADAM10 to rescue IM-stimulated shedding of EGF and BTC in Adam10−/− cells provided an opportunity to test how well chimera between ADAM10 and -17 (see A for a key to the constructs) are able to rescue shedding of the EGFR-ligands EGF or BTC in Adam10−/− cells. Only wild-type ADAM10 strongly enhanced shedding of EGF and BTC after addition of IM (D). Among the chimera tested here, only the deletion mutant A10b, which lacks the cytoplasmic domain of ADAM10, could partially rescue the IM induced shedding of EGF (~40%; D) and BTC (~25% E; see Supplemental Figure 2, A and B, for a comparison of the relative amounts of wild-type ADAM10 and -17 and of the chimera expressed in COS-7 cells).
The cytoplasmic domain of ADAM10 from several species (human, bovine, murine, and rat) contains a sequence resembling the calcium-independent IQ consensus binding site for calmodulin (IQXXXRXXXXR; for review, see (Bahler and Rhoads, 2002
), sequence in murine ADAM10: IQQPPRQRPRE). HA-tagged ADAM10 and a mutant in which the IQ sequence was mutated to AA (IQ > AA) were transfected into COS-7 cells, and the expressed proteins were visualized by Western blot analysis with anti-HA antibodies (F, lanes 1 and 3). Similar levels of the proform of wild-type ADAM10 and the IQ > AA mutant were detected; however, only very little mature ADAM10 IQ > AA was present compared with wild-type ADAM10. This suggests that the IQ sequence has a role in maturation of ADAM10, or in stabilizing the mature protein. Cell lysates expressing wild-type ADAM10 or the IQ > AA mutant were incubated with calmodulin/agarose beads, and the bound material was subjected to Western blot analysis with antibodies against the HA-tag. This showed that only the proform of wild-type ADAM10 as well as of the ADAM10 IQ > AA mutant bound to immobilized calmodulin (F, lanes 2 and 4), demonstrating that the IQ sequence is not essential for the interaction between calmodulin and pro-ADAM10. Further studies will be necessary to understand the involvement of the IQ sequence in the maturation or turnover of ADAM10 (please note that the effects of the IQ > AA mutation might be unrelated to any putative interaction of ADAM10 with calmodulin). Nevertheless, the availability of a mutant with impaired maturation or decreased stability of the mature protein provided an additional opportunity to test whether the decreased levels of mature ADAM10 IQ > AA compared with wild-type ADAM10 affect its response to calcium influx. Adam10
−/− cells were cotransfected with EGF or BTC and either wild-type ADAM10, the IQ > AA mutant, or an empty pcDNA3 vector as control. The decreased levels of mature ADAM10 IQ > AA did not affect the ability of this mutant to rescue IM stimulated shedding of EGF or BTC compared with wild-type ADAM10 (G), suggesting that other sequences in the cytoplasmic domain are responsible for the response of ADAM10 to calcium influx.
Calmodulin Inhibitor Stimulates Shedding of TGFα, HB-EGF, AR, and EPR in the Absence of ADAM17
As shown above and in A, PMA-stimulated shedding of TGFα, HB-EGF, AR, and EPR is abolished in Adam17−/− cells. Remarkably, shedding of these four EGFR-ligands could be strongly stimulated by TFP in Adam17−/− cells by an activity that was sensitive to BB94 (A). When we measured TFP stimulated shedding of TGFα and HB-EGF in cells lacking one or more of the widely expressed and catalytically active ADAMs that are candidate sheddases (ADAM8, -9, -10, -12, -15, and -19), a significant increase in shedding of TGFα and HB-EGF in response to TFP was seen in all cases (B), arguing against a major role of these ADAMs in TFP-stimulated shedding of TGFα and HB-EGF. Thus, different stimuli (calmodulin inhibition versus PMA) can activate distinct sheddases for these four EGFR-ligands in mouse embryonic cells.
Figure 7. TFP-stimulated shedding of TGFα, HB-EGF, AR, and EPR in the absence of ADAM17. (A) TGFα, HB-EGF, AR, and EPR are EGFR ligands that require ADAM17 for constitutive and PMA-stimulated shedding. These four EGFR-ligands were transfected into (more ...)
To learn more about the identity of the calcium-activated sheddase(s) for TGFα, we compared the inhibitor profile of TIMP1, -2, and -3 toward PMA-stimulated shedding of TGFα in wild-type mEF cells to the profile of the sheddase activated by calcium influx in Adam17−/− cells. Constitutive and PMA-stimulated shedding of TGFα was not blocked by up to 30 nM of TIMP1 and -2 or by 1 nM TIMP3, but it was inhibited between 50 and 70% by 30 nM TIMP3 compared with the inhibition with 1 μM BB94 (C). The calcium influx induced shedding of TGFα in Adam17−/− cells after addition of 2.5 μM IM was strongly inhibited by TIMP1, -2, and -3 at concentrations as low as 1 nM, as well as by 1 μM BB94, suggesting the involvement of an MMP that is sensitive to all three TIMPs (D).