Regulated ADAM17-dependent EGF family ligand
release by substrate-selecting signaling pathways
Michelle Danga,b,c,1, Nicole Armbrusterc,1, Miles A. Millerd, Efrain Cermenoa,d, Monika Hartmanne, George W. Bella,
David E. Rootf, Douglas A. Lauffenburgerd, Harvey F. Lodisha,b,d,2, and Andreas Herrlicha,c,2
aWhitehead Institute for Biomedical Research, Cambridge, MA 02142;bDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139;
cRenal Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115;dDepartment of Biological Engineering, Massachusetts Institute
of Technology, Cambridge, MA 02139;eLeibniz Institute for Age Research, Fritz Lipmann Institute, 07745 Jena, Germany; andfBroad Institute, Cambridge,
Contributed by Harvey F. Lodish, April 28, 2013 (sent for review December 6, 2012)
Ectodomain cleavage of cell-surface proteins by A disintegrin and
metalloproteinases (ADAMs) is highly regulated, and its dysregula-
tion has been linked to many diseases. ADAM10 and ADAM17
cleave most disease-relevant substrates. Broad-spectrum metallo-
protease inhibitors have failed clinically, and targeting the cleavage
of a specific substrate has remained impossible. It is therefore nec-
essary to identify signaling intermediates that determine substrate
specificity of cleavage. We show here that phorbol ester or angio-
tensin II-induced proteolytic release of EGF family members may
not require a significant increase in ADAM17 protease activity.
Rather, inducers activate a signaling pathway using PKC-α and
the PKC-regulated protein phosphatase 1 inhibitor 14D that is re-
quired for ADAM17 cleavage of TGF-α, heparin-binding EGF, and
amphiregulin. A second pathway involving PKC-δ is required for
neuregulin (NRG) cleavage, and, indeed, PKC-δ phosphorylation
of serine 286 in the NRG cytosolic domain is essential for induced
NRG cleavage. Thus, signaling-mediated substrate selection is
clearly distinct from regulation of enzyme activity, an important
mechanism that offers itself for application in disease.
epidermal growth factor receptor|transactivation
Ectodomain shedding generates many diverse bioactive cytokines
and growth factors, and governs important cellular processes in the
developing and adult organism, including the control of growth,
adhesion, and motility of cells (reviewed in refs. 1–3). EGF re-
ceptor activation generates signals for cell proliferation, migra-
tion, differentiation, or survival. The 12 EGF family members are
synthesized as cell surface transmembrane precursors. The active
growth factors are released by A disintegrin and metalloprotein-
ases (ADAMs) and activate specific heterodimeric EGF receptors
on the cell surface connected to diverse intracellular signaling
pathways (4,5).IncreasedsheddingofEGFligandshas beenlinked
to different clinical pathologic processes (6–10); hence, therapeutic
control of ligand release would be beneficial. Of the 12 functional
ADAMs encoded in the human genome (3) only two—ADAM10
and ADAM17—handle most of the numerous ADAM substrates,
in particular, the EGF ligands. However, broad-spectrum metal-
loprotease inhibitors tested for clinical use have failed as a result of
indiscriminate blockade of substrate cleavage, leading to clinical
side effects (11). Even recently developed selective ADAM inhib-
itors stillaffect the cleavage of many substrates (12). Modulation of
the release of specific ADAM substrates has been impossible to
date because it is unknown how cleavage specificity is regulated on
the molecular level. It is therefore necessary to identify key signals
that determine substrate specificity of cleavage.
Ectodomain cleavage is made specific by a number of in-
tracellular signals; e.g., by calcium influx, by activation of G pro-
tein-coupled receptors, and the release of diacylglycerol (reviewed
in refs. 3, 13). Several distinct mechanisms that modulate cleavage
on the level of ADAM17 have been described, including regula-
tion of ADAM17 expression, maturation, trafficking to the cell
surface (reviewed in ref. 13), and posttranslational modifications
he ectodomains of many cellsurfaceproteinsare shedfromthe
surface (i.e., “ectodomain shedding”) by metalloproteases.
on the ADAM17 ectodomain (14, 15) or its C terminus (16, 17).
However, modulation of activity of the relatively few available
ADAMs does not suffice to explain substrate-specific regulation
of cleavage (18, 19), and none of the referenced studies has
addressed how specificity of cleavage is achieved. Transgenic
overexpression of ADAM17 in mice does not lead to overactivity
of ADAM17 or increased ADAM17 substrate release, emphasiz-
ing the importance of posttranslational control of cleavage (20).
Most reports on induced shedding (reviewed in refs. 5, 21) have
only used monitoring of substrate cleavage as a surrogate measure
of protease activity. However, only few studies unequivocally
document induced changes of protease activity, and those were
small. A tight-binding ADAM17 inhibitor interacts with the cat-
alytic site of ADAM17 only after 12-O-tetradecanoylphorbol-13-
acetate (TPA; i.e., phorbol ester) stimulation (12), suggesting
regulation of the catalytic site. Another convincing example of
regulated enzyme activity has been based on observed effects of
oxidation on several putative disulphide bonds in the ADAM17
ectodomain that result in a structural change. This involves the
interaction with an extracellular redox regulator, protein disulfide
isomerase (PDI). PDI down-regulation enhanced TPA-induced
shedding of heparin-binding (HB) EGF, addition of exogenous
PDI decreased it, and PDI addition to recombinant ADAM17
reduced basal cleavage of a fluorescence resonance energy trans-
fer (FRET) peptide. These changes correlated with altered to-
pology of antibody epitopes outside of, but not within, the
catalytic domain (14).However, inducedHB-EGFcleavage could
ADAM17 via the altered topology outside of the catalytic domain
without requiring changes in protease activity. Neither study de-
termined protease activity independent of substrate cleavage, still
leaving us with uncertainty whether induced substrate cleavage
truly requires enhanced protease activity. By using stopped-flow
X-ray spectroscopy and other techniques, Solomon et al. showed
induced by the substrate before proteolysis (22). Novel exosite
inhibitors of ADAM17 activity that bind ADAM17 outside of the
catalytic site and likely interfere with the binding of glycosylated
moieties of the substrate have been developed (23). Both studies
further support regulation of proteolysis on the substrate level.
Here we identify pathway components that distinguish sub-
strates of ADAM17 and parse substrate selection from regula-
tion of protease activity.
Author contributions: H.F.L. and A.H. designed research; M.D., N.A., M.A.M., E.C., and A.H.
performed research; M.D., N.A., M.A.M., E.C., M.H., D.E.R., D.A.L., and A.H. contributed new
reagents/analytic tools; M.D., N.A., M.A.M., G.W.B., D.A.L., H.F.L., and A.H. analyzed data;
and H.F.L. and A.H. wrote the paper.
The authors declare no conflict of interest.
1M.D. and N.A. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or lodish@
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| June 11, 2013
| vol. 110
| no. 24www.pnas.org/cgi/doi/10.1073/pnas.1307478110
involved in cleavage regulation, but none clearly distinguished
whether protease or substrates were the target of regulation [e.g.,
PKC-e/TNF-α (44); PKC-δ/HB-EGF (45); PKC-δ and PKC-e/IL-
6R (46)]. In concordance with our study, PKC-α siRNA knock-
down blocks TPA-induced HB-EGF cleavage (47), and PKC-δ
and phosphorylated C-terminal serine residues regulate cleavage
of chicken NRG in neuronal cells (48).
In summary, we show that the regulatory proteins PKC-α,
PPP1R14D, and PKC-δ specifically affect the cleavage of only
select ADAM17 substrates without significantly affecting pro-
tease activity. This observation offers itself as a new avenue for
therapeutic intervention independent of the protease and pos-
sibly specific for a particular disease-causing substrate.
Materials and Methods
SI Materials and Methods describes materials, retrovirus/lentivirus pro-
duction and infection, IP, Western blotting and ELISAs, PP1 biochemical
assay, immunoprecipitated cell-surface ADAM17 protease assay, FACS, and
shRNA FACS screen.
PrAMA. For live-cell PrAMA, IPTG-induced MDA-MB-231 human breast cancer
cellswere seeded ina 384-well clear-bottomblack-walled plate. Thenextday,
samples were stimulated with internally quenched FRET substrates (5 μM)
alone or with TPA (1 μM) for 30 min. Substrates were also added to no-cell
(negative control) and trypsin (positive control). Fluorescence readings were
obtained every 10 min for 2 h at 37 °C.
ACKNOWLEDGMENTS. We thank the Massachusetts Institute of Technology
Swanson Biotechnology Center for help with image analysis; Issei Komuro
for providing HEK293T- angiotensin II type 1 receptor cells; Glenn Paradis
and Patti Wisniewski (FACS Facility), Kathleen Ottina (BL2plus Facility), and
Jen Grenier (Broad Institute) for their help; Prat Thiru and Bingbing Yuan for
help with biostatistical analysis; and Peter Herrlich for critical reading. This
work was supported by National Institute of Diabetes and Digestive and
Kidney Diseases Grants R00DK077731 and R01-CA96504.
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