1. 1 3
DOI 10.1007/s00018-014-1616-1 Cellular and Molecular Life Sciences
Cell. Mol. Life Sci. (2014) 71:3269–3279
Rev iew
Discoidin domain receptors: a proteomic portrait
Leo K. Iwai · Maciej T. Luczynski · Paul H. Huang
Received: 29 January 2014 / Revised: 18 March 2014 / Accepted: 20 March 2014 / Published online: 5 April 2014
© Springer Basel 2014
activated by collagen [ 1]. These receptors regulate a num -
ber of fundamental biological processes including cell
adhesion, migration, proliferation, and extracellular remod-
eling [ 1–3]. Dysre gulation of DDR function has been
implicated in se veral diseases such as ἀbrosis, atheroscle -
rosis, arthritis, and cancer [1, 4]. There are two members in
this RTK class with DDR1 primarily expressed in epithelial
cells and DDR2 in mesench ymal cells. Correspondingly,
induction of epithelial–mesenchymal transition results in a
decrease in DDR1 e xpression and a reciprocal increase in
DDR2 levels [5, 6]. Their domain organization is composed
of an e xtracellular re gion containing tw o N-terminal dis -
coidin domains, an e xtracellular juxtamembrane domain,
a transmembrane domain, an intracellular juxtamembrane
domain, a kinase domain follo wed by a short C-terminal
tail. In addition, while DDR1 binds to both ἀbrillar and
basement membrane collagen, DDR2 preferentially binds
to ἀbrillar collagens and collagen type X [7–9].
Unlike classical growth factor RTKs like the epider-
mal growth f actor receptor (E GFR) and platelet-deri ved
growth f actor receptor (PDGFR), DDR eng agement by
collagen leads to a slo w and sustained acti vation proἀle in
which tyrosine phosphorylation levels start to increase only
hours after lig and stimulation and remains persistent o ver
several days [ 10, 11]. DDR1 and DDR2 contain 15 and
14 c ytoplasmic tyrosine residues, respecti vely, which are
potential sites for protein phosphorylation and binding to
downstream signaling adaptors and effectors. W hile a small
number of targeted studies have attempted to identify these
effectors, data from these studies have failed to comprehen-
sively deἀne the components of DDR signaling networks.
Advances have largely been hampered by challenges asso-
ciated with DDR biology including the unique receptor
activation kinetics and the inherent multi-lig and nature
of collagen. In addition, while there are an ab undance of
Abstract The discoidin domain receptors (DDRs) are
collagen-binding receptor tyrosine kinases that ha ve been
implicated in a number of fundamental biological processes
ranging from growth and development to immunore gula-
tion. In this review, we examine how recent proteomic tech-
nologies ha ve enriched our understanding of DDR sign -
aling mechanisms. We provide an o verview on the use of
large-scale proteomic proἀling and chemical proteomics to
reveal novel insights into DDR therapeutics, signaling net -
works, and receptor crosstalk. A perspective of ho w prot-
eomics may be harnessed to answer outstanding fundamen-
tal questions including the dynamic re gulation of receptor
activation kinetics is presented. Collecti vely, these studies
present an emer ging molecular portrait of these unique
receptors and their functional role in health and disease.
Keywords Discoidin domain receptors · Collagen ·
Cell signaling · Proteomics · Mass spectrometry
Introduction
The discoidin domain receptors (DDRs) are a class of
receptor tyrosine kinases (R TKs) that bind to and are
L. K. Iwai and M.T. Luczynski contributed equally to this work.
L. K. Iwai · M. T. Luczynski · P. H. Huang ()
Protein Networks Team, Division of Cancer Biology, Institute
of Cancer Research, 237 Fulham Road, London SW 3 6JB, UK
e-mail: paul.huang@icr.ac.uk
Present Address:
L. K. Iwai
Laboratório E special de Toxinologia Aplicada/CeTICS, Instituto
Butantan, Av V ital Brasil 1500, São Paulo 05503‑000, Brazil

2. 3270 L. K. Iwai et al.
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studies that pro vide in vitro e vidence of the signaling and
functional properties of the DDRs in cell culture, there is
less data supporting the role of the DDRs as acti ve signal-
ing molecules in tissues and organs.
In the past 5 years, proteomics has undoubtedly emerged
as a po werful tool to further our understanding of DDR
biology. In this perspective, we will provide an overview
of how this technology has produced a molecular portrait
of DDR signaling mechanisms in human health and dis -
ease (outlined in Fig. 1). We further elaborate on the use
of proteomics to enrich our understanding of DDR struc -
ture–activity relationships as well as the reprogramming of
signaling in response to therapeutic intervention. Finally,
we offer a prospective view of the potential of proteomics
to pro vide future insights into une xplored areas of DDR
biology.
Proteomic proἀling of DDR activity in cancer
Phosphorylation is a re versible and highly dynamic signal
transduction mechanism that plays an important role in the
regulation of a myriad of cellular processes. The impor -
tance of this post-translational modiἀcation is e xempliἀed
by aberrant global phosphorylation e vents that ha ve been
reported in multiple pathologies such as cancer , and meta-
bolic and autoimmune diseases. In particular, dysregulation
of protein phosphorylation has been particularly well stud -
ied in cancer where multiple oncogenes and tumor suppres-
sors lie in protein phosphorylation pathw ays that directly
inḀuence cancer progression [12]. It is thus not surprising
that drugs tar geting protein kinases represent the lar gest
class of compounds currently a vailable to treat this disease
[13]. Le veraging on the dependenc y of cancer cells for
oncogenic signaling pathw ays, these kinase-speciἀc tar -
geted therapies ha ve led to dramatic clinical responses in
subsets of cancer patients [ 14–16]. Furthermore, monitor -
ing components in kinase signaling pathw ays are increas -
ingly being used as biomark ers for stratiἀcation of patient
response to therapies as well as recruitment criteria for clin-
ical trials. These developments in clinical practice to wards
personalized medicine ha ve made it necessary not only to
better understand the dynamic nature of kinase signaling
networks but also to de velop improved methods for moni -
toring phosphorylation levels in clinical specimens.
Ampliἀcation and mutations in the DDRs ha ve been
reported in multiple cancer types, including breast, o var-
ian, brain, and lung tumors [17–21] (a complete review of
the role of DDRs in cancer can be found in [3]). Previous
studies in E GFR-positive cancer patients suggest that total
Fig. 1 A timeline of proteomic studies that have informed our current understanding of discoidin domain receptor biology

3. 3271Proteomics of discoidin domain receptors
1 3
E GFR protein levels is a poor predictor of patient response
to E GFR kinase inhibitors. In contrast, a high phospho-
E GFR level, the active form of the receptor, is a good prog-
nostic indicator of sensiti vity to E GFR-targeted therap y
[22, 23]. Along these lines, it w ould be vital to determine
if DDRs are in f act active in tumors where protein o verex-
pression/mutation is observ ed and if so, do the y represent
good biomarkers for patient response to kinase inhibitors.
Despite the functional importance of the DDR acti vity in
vitro, for about a decade follo wing the disco very of the
DDRs, there were no studies that conclusi vely demon -
strated that these receptors were indeed phosphorylated or
active in vivo. This paucity of e vidence was largely due to
the lack of phospho-speciἀc reagents (e.g., antibodies) tar -
geting this class of receptors.
The introduction of unbiased phosphoproteomic proἀl -
ing by mass spectrometry (MS) rapidly o vercame these
technical challenges. Rik ova et al. [ 24] pro vided the ἀrst
study to demonstrate the scale of DDR phosphorylation in
vivo. The authors performed a lar ge-scale screen of tyros -
ine phosphorylation levels in 150 non-small cell lung carci -
noma (NSCLC) tumors and 41 cell lines. They were able to
identify more than 2,700 tyrosine-phosphorylated proteins,
which included over 50 different tyrosine kinases. This study
established that signiἀcant le vels of phosphorylated DDR1
and 2 were present in lung cancer . A comparison of phos -
phorylated tyrosine kinase proἀles in tumors versus cell lines
raised an interesting observ ation. W hile the analysis of cell
lines revealed the activation of common NSCLC targets such
as E GFR, c-Met, and E phA2 [ 25–27], the patient-deri ved
tumor proἀles sho wed that the DDRs w as by f ar the most
commonly activated tyrosine kinase class in vivo, accounting
for more than a third of the tumors analyzed. Importantly ,
very few DDR1 and no DDR2 phosphorylation e vents were
found in the cell lines. This data highlights the limited utility
of NSCLC cell lines in recapitulating in vivo tyrosine kinase
signaling events observed in patients. Furthermore, the pres -
ence of DDR acti vation only in clinical specimens suggests
that the lung tumor microen vironment, which contains high
levels of collagen [28], is critical for modulating signaling
networks in vivo. The same group performed a subsequent
screen of cholangiocarcinoma tumors from 23 patients [ 29].
The unique aspect of this study was the comparison of tumor
specimens with para-tumor (normal) tissues. Consistent with
the NSCLC study, high levels of activated DDRs was found
in more than 70 % of the patients studied, conἀrming that
these receptors are prevalent in multiple tumor types.
More recently, a phosphoproteomic analysis of ten cell
lines and three human tumors representing different forms
of the sarcoma identiἀed tyrosine phosphorylation of
DDR2 in 9/10 cell lines and 1/3 tumors [ 30]. These ἀnd-
ings are congruent with the restricted e xpression of DDR2
in cells of mesench ymal origin. Furthermore, sarcoma
cell lines are kno wn to secrete a lar ge amount of collagen,
which represents a source of autocrine lig and for the acti -
vation of DDR2 [ 10, 31, 32]. It should be noted that all of
the described studies above also identiἀed a large network
of other RTKs that are simultaneously co-acti vated in can-
cer cell lines and tumors that poses several important ques-
tions [33, 34]. Firstly, why do cells simultaneously activate
multiple tyrosine kinases in the context of cancer but not in
normal tissues? The second question is ho w does one de-
convolute these complex RTK networks and resolve which
downstream signals are speciἀc to particular R TKs such as
the DDRs? Finally, is there bi-directional crosstalk between
the DDRs and other tyrosine kinases embedded within
these RTK co-activation networks?
A recent proteomic proἀling in glioblastoma sheds some
light on the latter question. In this study , eight patient-
derived glioblastoma tumor x enografts e xpressing wild-
type or mutant forms of E GFR were subjected to both
quantitative proteomic and phosphoproteomic analysis
[35]. One of the intriguing ἀndings of this study w as that
well-established interactors of E GFR such as SHC, STAT3,
Cbl, Gab1, and PLC γ1 did not cluster with E GFR phos -
phorylation. In contrast, phosphorylation sites on the acti -
vation loop of DDR1 strongly correlated with the majority
of E GFR tyrosine phosphosites across all the tumors stud -
ied. This clustering analysis suggests that in GBM tumors,
crosstalk between E GFR and DDR1 is lik ely to be a sig -
niἀcant event, and that the resultant do wnstream pathways
from this interaction may be distinct from E GFR speciἀc
signaling networks. Taking an in vitro approach, our group
explored the potential for crosstalk between the insulin
receptor (IR) and DDR2. H E K293 cells e xpressing both
DDR2 and IR w as subjected to stimulation with insulin
and/or collagen [36]. Using iTRAQ labeling methodol-
ogy, we sho wed that insulin enhanced collagen-mediated
DDR2 activation. We also identiἀed SHIP-2 and SGK269
as potential do wnstream effectors that arise from the inte -
grated activation of DDR2 and IR. The exact mechanism
by which IR and E GFR interact with the DDRs is still
uncertain. Ho wever, the observ ed crosstalk between the
DDRs and other R TKs may represent a means for signal
diversiἀcation of collagen receptor signaling to achie ve
robustness despite e xposure to e xogenous insults, includ -
ing tar geted therap y [ 37]. Further in vestigation into the
extent and biological consequence of DDR-R TK crosstalk
is needed to resolve these questions.
The identiἀcation of acti ve DDRs in multiple cancers
conἀrms that DDR acti vation occurs in vi vo and may play
an important role in the tumor initiation and/or progres -
sion. These developments have led the w ay for the search
of small molecule inhibitors that would target the DDRs
and disrupt its acti vity and resultant signaling pathw ays in
cancer.

4. 3272 L. K. Iwai et al.
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Chemical proteomics and the DDRs
In order to better characterize the mode of action of kinase
inhibitors, MS-based chemical proteomics has successfully
been used to identify the tar get(s) of these small molecules
and their effects on tumor signaling netw orks. In a typical
chemical proteomics w orkḀow, an immobilized chemi -
cal probe or drug of interest is e xposed to a protein e xtract
and target proteins that bind to these probes are then iden -
tiἀed and quantiἀed by disco very-based MS proteomics
(Fig. 2a). This approach has been ef fective in identifying
the target proἀle of the multi-kinase inhibitors de veloped
for treatment of chronic myelogenous leuk emia (CML).
Kinase inhibitors such as dasatinib, nilotinib, and imatinib,
which tar get the constituti vely acti ve BCR-ABL fusion
oncoprotein, have been approved for the treatment of CML
since 2003. Imatinib is the ἀrst-line treatment for CML
positive for the Philadelphia chromosome, while dasatinib
and nilotinib are currently being used for imatinib-intol-
erant and -resistant CML patients, although the y still f ail
in patients harboring the g atekeeper mutation at T315I in
BCR-ABL [38].
Despite being rationally designed to target BCR-ABL,
several chemical proteomic studies ha ve since sho wn that
these drugs also inhibit other kinases including c-Src,
c-KIT, PDGFR, and the DDRs [ 39–41]. In f act, Imatinib
is no w appro ved for adv anced g astrointestinal stromal
tumor (GIST) patients with c-KIT e xpression as well as
other malignancies with PDGFR translocations [42–44].
Through an afἀnity-based chemical proteomic approach
using kinobeads (an immobilized non-selecti ve kinase
inhibitor matrix) in K562 CML cells, Bantscheff et al. [39]
identiἀed o ver 150 protein kinases that were capable of
binding to imatinib and dasatinib with varying afἀnities. In
addition to conἀrming the mode of action of these drugs
on the ABL and SRC f amily kinases, the study also deter -
mined that both DDR1 and 2 bound to these compounds
with nanomolar afἀnity . The authors v alidated these ἀnd -
ing using in vitro kinase assays, which demonstrated that
DDR1 is a bona ἀde tar get of imatinib . A further study
by Rix et al. [41] on all three compounds immobilized to
a Sepharose matrix and incubated with K562 and CML
primary cell lysates sho wed that nilotinib and dasatinib
also bound strongly to DDR1. These studies corroborate
sequence alignment and molecular modeling experiments
which show striking similarities in the ATP-binding pocket
of the DDRs and other identiἀed tar get kinases such as
ABL and c-KIT [ 45]. Taken together, these chemical pro -
teomic studies demonstrate that these three compounds
are candidate drugs for malignancies dri ven by oncogenic
DDRs. Consistent with this idea, lung cancer cell lines with
oncogenic DDR2 mutations are susceptible to dasatinib
treatment [19].
Chemical proteomic analysis of cancer therapeutics
is not limited to kinase inhibitors. HSP90 is a chaper -
one protein that assists in the folding and stabilization of
multiple client proteins, including well-established onco -
proteins such as B-RAF , E RBB2, CDK4, and BCR-ABL
[46]. The essential function of HSP90 in maintaining
oncoprotein expression provides a therapeutic rationale
for the use of HSP90 inhibitors in cancer therap y. Using
stable isotope labeling with amino acids in cell culture
(SILAC) methodology , Sharma et al. [ 47] treated HeLa
cervical cancer cells with the HSP90 inhibitor , 17-DMAG
[17-(dimethylaminoethylamino)-17-demethoxygeldan-
amycin] and monitored changes in 6,000 proteins and
4,000 phosphopeptides. This study sho wed that inhibition
of HSP90 results in pleiotropic ef fects on multiple cellu -
lar processes including DNA metabolism, protein synthesis
and degradation, cell cycle, and apoptosis.
In another study performed by W u et al. [48], an inte -
grated proteomics strate gy comprising multiple dif fer-
ent techniques was used to study the ef fects of the HSP90
inhibitor geldanamycin on four dif ferent cancer cell lines.
These techniques comprise SILAC quantiἀcation and
chemical precipitation of kinases using kinobeads, HSP90
immunoprecipitation, and immobilized geldanamycin on
Sepharose beads. They identiἀed o ver 1,600 proteins that
were signiἀcantly altered upon drug treatment. Of these,
98 were protein kinases that were do wn-regulated and
classiἀed as HSP90 clients. DDR1 was among this list of
kinases and a protein turno ver-rate analysis using pulsed
SILAC e xperiments at 6, 12, and 24 h combined with
kinobeads enrichment sho wed that, upon drug treatment,
DDR1 underwent a more rapid de gradation compared
to E GFR, E RK1, or E PHA2. This observ ation pro vides
important pharmacokinetic information for drug response
where targeting HSP90 w ould lead to a more rapid ef fect
on DDR1 compared to other kinases and client proteins.
Fig. 2 Proteomic strate gies used to elucidate discoidin domain
receptor signaling netw orks and chemical interactions. a Schematic
of general chemical proteomic approach for identiἀcation of drug/
probe-binding target proteins. Cell or tissue lysate is incubated with
immobilized probes (e.g., kinobeads) or drug to isolate interacting
proteins. Subsequent elution and mass spectrometry analysis result
in the identiἀcation of probe/drug tar gets. b Different methodologies
used for the identiἀcation of DDR signaling. (i) Proἀling of activated/
phosphorylated kinases in cancer cell lines and tumors using global
phosphoproteomic approaches. ( ii) Catalog ef fects of drug treatment
(kinase or HSP90 inhibitors) in cancer cell lines on cellular pro -
teome and phosphoproteome can inform potential no vel combination
regimens for disease. ( iii) Treatment of DDR-e xpressing cells with
phosphatase inhibitor results in global upre gulation of cellular phos -
phorylation. Subsequent DDR immunoprecipitation and mass spec-
trometry analysis identiἀes phosphorylation-mediated protein inter -
actions. (iv) Stimulation of DDRs with exogenous collagen leads to
the activation of receptor -speciἀc signaling netw orks, which can be
proἀled with global phosphoproteomics
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5. 3273Proteomics of discoidin domain receptors
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A
B

6. 3274 L. K. Iwai et al.
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Another study employing a similar approach also identiἀed
DDR2 as client protein of HSP90 [ 49]. Collectively, these
data demonstrate that the DDRs are major client proteins
of HSP90, and HSP90 inhibitors may ha ve utility ag ainst
tumors that are DDR positive.
These studies also raise an interesting question as to
whether a combination approach of using both kinase and
HSP90 inhibitors would be more effective in eliminating
DDR oncogenic signaling compared to either drug alone
(Fig. 2b). This combination would be particularly important
since gatekeeper mutants of DDRs are resistant to dasat -
inib inhibition [ 19, 30, 45, 50]. W hile such DDR muta -
tions have yet to be reported in patients, based on reports
in other oncogenic kinases [ 51, 52], it is lik ely that g ate-
keeper mutations will arise with prolonged use of imatinib
or dasatinib. Indeed, in vitro modeling of dasatinib resist -
ance in lung cancer cell lines results in the de velopment of
the T654I gatekeeper mutation in DDR2 as a mechanism
of acquired resistance [53]. In a recent study of HSP90 and
its recruitment to protein kinases via CDC37, Polier et al.
[54] analyzed the mode of action of dif ferent ATP-compet-
itive kinase inhibitors on HSP90-CDC37-kinase chaper -
one complex formation. Their study revealed a dual mode
of action for this class of inhibitors. These drugs not only
acted as con ventional ATP-competitive kinase inhibitors
by competing for ATP binding b ut also serv ed as antago -
nists depriving client kinase recruitment to the HSP90 and
CDC37 chaperone system f acilitating kinase de gradation
by proteasomes. These data suggest that a combination
with HSP90 inhibition may boost the client protein degra-
dation capability of kinase inhibitors, which will enhance
therapeutic efἀcac y and reduce the lik elihood of de vel-
oping drug-resistant gatekeeper mutations. Furthermore,
multi-target kinase inhibitors, such as dasatinib described
above, inhibit the DDRs as an unanticipated “off-target”
effect. More recently, DDR selective inhibitors, which are
capable of inhibiting cancer cell proliferation and migra -
tion, have been identiἀed by several groups [55–59]. These
compounds have the potential to progress to become potent
anti-cancer agents in DDR-driven cancers.
Cellular signaling is a dynamic process that constantly
adapts to both endogenous and e xogenous stimuli. Another
interesting facet of DDR biology is the ability of tumor cells
to modulate receptor expression levels in response to thera-
peutic challenge. This “kinase reprogramming” phenom -
enon was studied in depth by Duncan et al. using a chemi -
cal proteomics approach. E mploying multiple xed kinase
inhibitor beads and mass spectrometry (MIB/MS) to enrich
for kinases in triple-ne gative breast cancer cells, treatment
with ME K inhibitors AZD6244 and U0126 resulted in sig -
niἀcant reprogramming of the kinome induced by the pro -
teasomal c-Myc de gradation [ 60]. Initial treatment with
the drugs inhibited cell gro wth and elevated RNA levels of
RTKs including DDR1 and DDR2. Continuous drug e xpo-
sure o ver 30 days generated therap y-resistant cells with
increased protein levels of ME K2, VE GFR2, and PDGFRβ
and elevated tyrosine phosphorylation of ME K, AXL, RAF,
and AKT. The individual depletion of some of the repro -
grammed R TKs such as DDR1, DDR2, AXL, VE GFR2,
and PDGFR β using siRN A in combination with U0126
were able restore gro wth arrest. This data indicates that a
single knockdown of DDR1 or DDR2 in the presence of a
ME K inhibitor is able to kill triple-negative breast cancer
cells. Interestingly , combination therap y using AZD6244
with a low dose of RTK inhibitors (sorafenib, a PDGFRα/β,
VE GFR, DDR1/2, and RAF inhibitor; and foretinib, a cMet
and VE GFR inhibitor), which were inef fective as single
agents, inhibited phosphorylation of multiple R TKs and
efἀciently arrested growth of SUM159 triple-negative breast
cancer cells. Importantly , in vi vo experiments using breast
cancer C3Tag mouse model treated orally with AZD6244
also showed kinome reprogramming with increased expres-
sion of M E K2, E RK1, PDGFRβ, and DDR1 recapitulating
the reprogramming response observ ed in vitro. Treatment
of C3Tag mice with AZD6244 and Sorafenib led to tumor
growth arrest, increased apoptosis, and tumor regression
in the majority of mice. These ἀndings show that a deἀned
combination therapy based on the a priori knowledge of the
kinome pattern of resistance is ef fective in producing sig -
niἀcant therapeutic beneἀt.
Although the role of DDR in cancer is still poorly
characterized, MS-based chemical proteomics has been
exploited to monitor changes in the composition of the
cancer kinome, including DDR1 and DDR2, upon M E K
inhibitor treatment. Cancer cells circumv ent the inhibition
of oncogenic signaling pathways by upregulating the DDRs
and additional survival routes, highlighting the importance
of this class of receptors in conferring resistance to kinase
inhibitor therapy. On a more global scale, multiple cancer
types are driven by Myc activation [61] and this study sug-
gests that upre gulation of DDRs in the conte xt of a R TK
co-activation netw ork may be a general mechanism by
which cancer cells evade elimination of the Myc pathway.
DDR signaling networks
Perhaps the most substantial contrib ution of proteomics to
DDR biology is the lar ge-scale elucidation of its signaling
Fig. 3 Reported intracellular protein–protein interactions and do wn-
stream signaling components of a DDR1 and b DDR2. The ἀgure
depicts known tyrosine phosphorylation sites ( black squares) in the
cytoplasmic domains of both receptors and the do wnstream effector
proteins (white squares) that have been shown to either interact with
or be phosphorylated by the DDRs. Associated references for the
reported interactions are indicated in parentheses
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7. 3275Proteomics of discoidin domain receptors
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8. 3276 L. K. Iwai et al.
1 3
networks. There is a wealth of kno wledge of the collagen-
binding properties of the DDRs [ 2]; in contrast, the sign -
aling pathw ays acti vated by these receptors are lar gely
unknown. A decade of biochemical studies on the DDRs
has identiἀed a handful of signaling ef fectors including
SHC, CSK, PI3K, SHP2, R UNX2, and NCK1/2 [ 11, 62–
67]. E merging data from proteomics studies ha ve revealed
complex interactions in DDR do wnstream signaling net -
works. F or instance, in order to characterize the global
landscape of possible DDR1 interacting proteins, Lemeer
et al. emplo yed a strate gy where DDR1-o verexpressing
glioblastoma cells were treated with a tyrosine phosphatase
inhibitor pervanadate, and subjected to co-immunoprecip -
itation with anti-DDR1 antibodies (Fig. 2b) [ 68]. Utiliz -
ing quantitative MS based on SILA C methodology, more
than 30 proximal signaling proteins interacting with DDR1
were identiἀed. These proteins include some pre viously
described interactors b ut also highlighted ne w interactions
such as GRB2, E PHA2, RASGAP, SHIP1, SHIP2, and
STATs. Furthermore, in order to identify the speciἀc tyros -
ine phosphorylation residues in DDR1 that were responsi -
ble for mediating these interactions, the authors performed
phosphopeptide pull-down experiments incubating placenta
lysates with all 15 DDR1 tyrosine phosphorylated synthetic
peptides immobilized on Sepharose beads. They addition-
ally validated the identiἀed interactors by performing quan-
titative competition-binding experiments with phosphoryl-
ated peptides and non-phosphorylated controls using TMT
6-plex chemical labeling follo wed by MS analysis. RAS -
GAP was shown to interact with Y484, Y543, and Y586
while STAT1a/b, STAT3, and ST AT5b with the juxtam -
embrane Y703 and the acti vation loop Y796 sites. In addi -
tion, they found that both activators (PI3K was shown to
bind to pY881) and ne gative regulators (SHIP1 and SHIP2
were shown to bind to pY740) of the PI3K pathw ay were
arranged in close proximity on DDR1, indicating a poten -
tial mechanism for compartmentalized pathw ay regulation.
Taken together, this work characterizes the cellular interac-
tome of DDR1 and pro vides a roadmap for future mecha -
nistic studies on the roles of these effectors on DDR1 sign-
aling and biology . The binding partners to the DDR1 and
its speciἀc phosphorylation sites are summarized in Fig. 3a
[11, 48, 62–65, 68–77].
W hile the interactome study pro vides a catalogue of all
possible DDR1 interactions, it may not accurately repre -
sent the situation in cells since both temporal and spatial
effects are o verlooked in these e xperiments. Furthermore,
it is unclear as to which of the tyrosine residues in DDR1
are actually phosphorylated in cells in response to collagen
activation since the global tyrosine phosphatase inhibitor
pervanadate was used in this study. Using a complementary
strategy to identify DDR signaling effectors, our group has
performed an iTRAQ-based quantitative phosphoproteomic
analyses in DDR2-overexpressing cells to map the tempo -
ral activation of signaling networks in response to collagen
stimulation over the course of 24 h (Fig. 2b) [78]. We ἀnd
that speciἀc phosphorylation sites on the DDR2 recep -
tor were dif ferentially re gulated upon collagen eng age-
ment in cells. For instance, the activation loop sites Y736
and Y740 as well as the no vel Y684 and Y813 sites pre -
sented a delayed acti vation with maximal phosphorylation
at 24 h, while Y481 in the juxtamembrane re gion of the
receptor displayed a distinct constitutively phosphorylated
proἀle. These ἀndings support the idea that the re gulation
of RTK signaling is achie ved through site-speciἀc dif fer-
ences in both magnitude and kinetics of receptor phospho -
rylation [79]. Bioinformatic analysis identiἀed 45 proteins
including SHP-2, SHIP-2, PIK3C2A, E RK1, and PLCL2
that clustered strongly with DDR2, implicating these pro -
teins as downstream effectors of DDR2 signaling netw ork.
Importantly, these phosphorylation e vents were sho wn to
be speciἀc to DDR2 activation and independent of the col-
lagen-binding inte grins. These DDR2 signaling ef fectors
are summarized in Fig. 3b [36, 66, 67, 80–82].
Perspectives
An outstanding fundamental question in DDR biology is
how collagen binding promotes the delayed and sustained
kinetics of DDR acti vation. DDRs e xist as preformed
dimers [83–85] that under go a series of autophosphoryla -
tion e vents upon lig and eng agement. Understanding the
sequential order of receptor phosphorylation may shed
light on the molecular basis of the unique kinetic proἀle of
the DDRs. W hile proteomic studies have shown that differ-
ent sites on the DDRs have distinct activation kinetics [78],
the initial phosphorylation and subsequent dephosphoryla -
tion dynamics of these receptors are unknown. MS in com-
bination with quenching is a useful tool for elucidating the
site-speciἀc order of such R TK phosphorylation e vents.
E mploying this approach, Furdui et al. ele gantly sho wed
that ἀbroblast growth factor receptor 1 (FGFR1) autophos -
phorylation is dictated by a precisely ordered sequence of
events [79]. In this method, FGFR1 and ATP are mixed and
quenched prior to sequencing by MS, which allo wed the
authors to monitor each distinct tyrosine phosphorylation
site on FGFR1 re vealing a unique receptor acti vation pro-
ἀle. They showed that initially , asymmetric dimer forma -
tion of the receptor f acilitates the transphosphorylation of
an activation loop tyrosine residue, followed by subsequent
phosphorylation of other docking site tyrosines. In the last
step, a dif ferent tyrosine residue in the acti vation loop is
phosphorylated, increasing the overall kinase activity of the
receptor, f acilitating do wnstream signal transduction [ 79,
86]. A similar characterization of temporal DDR receptor

9. 3277Proteomics of discoidin domain receptors
1 3
phosphorylation will identify the key tyrosine residues that
are critical for kinase activity and the mechanisms that gov-
ern the observed delayed and sustained activation kinetics.
The DDRs and their lig ands are subjected to dynamic
regulation in vivo [87]. For instance, receptor acti vation is
modulated by matrix metalloproteinases either directly by
cleavage of its e xtracellular domain or indirectly through
degradation of the collagen matrix [ 88]. In addition, ligand
competition by matrisomal proteins such as SPARC and
crosstalk with other transmembrane receptors including
the integrins may also inḀuence DDR acti vation and con -
sequent downstream signaling [7, 89, 90]. E lucidating this
complex biology will require the acquisition of dynamic
signaling maps that detail netw ork changes in response to
cellular perturbation. Building on previous DDR interac-
tome studies, tar geted proteomic approaches such as mul -
tiple reaction monitoring (MRM) offers the powerful capa-
bility of reproducible acquisition of dynamic data across
multiple conditions, replicates and protein species. A recent
example of this technology by Zheng et al. [ 91] showed
that the SHC1 adaptor protein is a highly dynamic regula-
tor of E GFR signaling netw orks. In this study , ἀbroblasts
were stimulated with E GF and dif ferent phosphorylation
sites within SHC1 as well as 41 dif ferent SHC1-protein
binding partners were monitored o ver 16 time points by
MRM. This high-resolution analysis of the assembly/dis -
assembly dynamics of protein-binding partners to SHC1
revealed a stage-speciἀc function of this scaf fold protein.
Upon initial activation by E GF, proteins directing cell divi-
sion and survi val were enriched, switching to an interme -
diate stage involving functions like vesicle trafἀcking, and
ἀnally progressing to a ἀnal stage characterized by proteins
involved on cytoskeletal reorganization and downregulation
of the initial cell di vision signals. Applying this strate gy
to the dynamic interactome and signaling netw orks of the
DDRs may provide a molecular explanation of the intrigu -
ing activation kinetics of the DDRs and the functional rel -
evance of its slow and prolonged signaling network proἀle.
Conclusions
Proteomics has contributed to the unraveling of DDR biol-
ogy and their functional impact in health and disease. W hile
biochemical, structural, genetic, and physiological studies
performed by multiple groups in last decade have undoubt-
edly led to a better understanding of their biological prop -
erties, there is a still much to learn about these atypical
RTKs. In an era of collaborati ve science and inte grative
biology, we anticipate that “Omic” technologies (including
proteomics) in combination with classical approaches hold
the exciting potential to pro vide a comprehensive molecu-
lar portrait of these receptors.
Acknowledgments The work in the authors’ laboratory is funded
by the Wellcome Trust (W T089028) and the Biotechnology and Bio -
logical Sciences Research Council (BB/I014276/1).
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