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,3
4
Departments of
* Physiology and Biophysics and
Anatomy and Neurobiology, College of Medicine, University of California, Irvine, California 92697-4560, USA; and
Centro di Biotechnologie Avanzate, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
4Correspondence: Department of Physiology and Biophysics, College of Medicine, Med Sci I D238, University of California, Irvine, CA 92697, USA. E-mail: rablab{at}uci.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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through the
carboxyl-terminal tyrosine of the TrkA domain (Y785 in TrkA residue
numbering). Mutation of this site (Y
F) eliminated PLC activation
(indicating there are no other cryptic binding sites for PLC in the
DDR1 sequences) and markedly reduced the differentiative activity of
the receptor. This is in contrast to TrkA (or PDGFRß/TrkA chimeras),
where ablation of this pathway has no notable effect on PC12 cell
morphogenic responses. Thus, the activation of FRS2 and Shc (leading to
MAPK activation) is weaker in the DDR1/TrkA chimeras than in TrkA
alone, and the PLC contribution becomes essential for full response.
Nonetheless, both DDR1 JM regions contain potentially usable signaling
sites, albeit they apparently are not activated directly in DDR1 (or
DDR1 chimeras) in a ligand-dependent fashion. These findings suggest
that the DDR1 receptors do have signaling capacity but may require
additional components or altered conditions to fully activate their
kinase domains and/or sustain the activation of the JM sites.—Foehr,
E. D., Tatavos, A., Tanabe, E., Raffioni, S., Goetz, S., DiMarco,
E., De Luca, M., Bradshaw, R. A. Discoidin domain receptor 1
(DDR1) signaling in PC12 cells: activation of juxtamembrane domains in
PDGFR/DDR/TrkA chimeric receptors.
Key Words: tyrosine kinase • collagen receptor • signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. These receptors generally consist of an
extracellular ligand binding domain (ED), usually characterized by
various binding motifs, a single pass transmembrane (TM) segment, and a
cytoplasmic tyrosine kinase tethered by a juxtamembrane (JM)
region and flanked by a carboxyl-terminal tail, each of variable
length. Ligand binding induces autophosphorylation, which is a critical
step for activation of the kinase and the subsequent recruitment of
adapters/effectors that propagate signal transduction (2)
.
Tyrosine residues that become phosphorylated on the intracellular
domain (ID) of RTKs during this process can act as binding sites for
these proteins although they do not account for all such interactions.
The bound proteins in turn are also phosphorylated, leading to direct
production of a signal, such as with phospholipase C (PLC), or
the formation of a complex that may have multiple components, such as
the Shc-Grb2-SOS-Ras assembly, which activate downstream cascades,
involving other kinases/phosphatases. The ligands for these receptors
are mainly soluble messengers, i.e., hormones and growth factors. In
addition, the ligand-activated receptor complex is characterized by a
dimeric structure and individual receptors can induce several pathways
simultaneously. The initial responses are also essentially
instantaneous with receptor autophosphorylation detectable at the
earliest time points after ligand addition (for review, see ref
3
).
The discoidin domain receptors (DDR) are organizationally similar to
other RTKs (Fig. 1
) but are also characterized by a number of distinguishing differences.
In addition to the discoidin-like structure that characterizes the
ectodomain, they have JM sequences that are significantly longer than
other RTKs; the DDR1 isoforms occur as two splice variants, where a 37
residue insert is added to the already long JM region to make an even
longer segment (designated DDR1b). In addition, the amino-terminal
segment of the kinase domain (KD) is altered such that the G-X-G-X-X-G
signature sequence is displaced by 12 residues relative to other RTK
KDs. Recently, several types of collagen were found to activate the
DDRs, either in soluble or precipitated form, in a fashion consistent
with ligand binding (5
, 6)
. However, the responses
induced were extraordinarily slow, requiring 90–120 min to produce
maximal autophosphorylation and were sustained for a prolonged period
(~18 h). DDR1b contains a consensus Shc binding site, L-S-N-P-A-Y
(DDR1 does not), which is phosphorylated after extensive exposure to
collagen (6)
. However, it apparently does not activate
MAPK, and no other signaling responses have been identified (6
, 7)
.
|
The DDR family is broadly expressed physiologically and their genes
have been cloned from many species, accounting for the profusion of
designations: DDR (now DDR1 and 2), MCK-10, CCK2, trkE, NTRK4, EDDR,
RTK6, Cak, Ptk-3, NEP, TKT, and Tyro 10 (8
9
10
11
12
13
14
15
16)
. In early
development, DDR1 is found predominantly in neuroectoderm, whereas in
adult tissues it occurs in the epithelial cells of brain, lung, kidney,
and the gastrointestinal tract (6
, 8
9
10
11
12
13
, 16)
. DDR2 is
also found in brain, muscle, heart, and connective tissue (6
, 8
, 11
, 14
15
16)
. There is significant expression in a variety of
tumor cells, including modest levels in PC12 (8
, 10
, 11
, 16
, 17)
.
The physiological role of the DDR family is still obscure. The
discoidin-like domain in the extracellular portion of these receptors
suggested they might be involved in cell–cell interactions since
discoidin, which occurs in the cellular slime mold D.
discoideum, is a lectin (18)
. Indeed, at least in the
case of DDR2, the carbohydrate that is covalently attached to the
collagen appears to contribute to the ligand binding (6)
.
However, given the nature of the identified ligands, it seems more
likely that the DDRs interact with the extracellular matrix and may
work in concert with other molecules, such as the integrins, which also
bind collagen.
Studies to better understand receptor function in cells have often used
site-directed mutagenesis and overexpression of the RTK of interest.
However, this approach can be hampered by the fact that stimulating
endogenous RTK signals is unavoidable if the same receptor (or a
related family member) is endogenously expressed. To circumvent this
problem, chimeric receptors, in which the ED domain of a receptor not
normally expressed in the cells under investigation is fused to the
TM/ID of the RTK of interest, can be used. This has been accomplished
in PC12 cells by linking the binding domain of the PDGFR and the TM/ID
of TrkA or FGFR1 (PTR and PFR1) to identify their salient signaling
mechanisms (19
, 20)
. PC12 cells, stably expressing these
chimeras, differentiate in a ligand-dependent manner, and a variety of
mutagenesis experiments have helped to define the signaling pathways
used by both receptor types (19; E. D. Foehr and R. A.
Bradshaw, unpublished results).
In view of the fact that native DDRs have not yet been shown to generate ligand-responsive intracellular signals in the same fashion as other RTKs, PDGFR/DDR chimeras (similar to PTR and PFR1) were constructed and tested in stably transfected PC12 cells. However, these also failed to produce detectable signaling events or cellular responses. This is likely due either to a lack of signaling capacity or a weak/inactive KD (or both). To resolve this question, the DDR1 KD was replaced by that of TrkA in the PDGFR/DDR1 chimera. At the same time, the DDR1 (and 1b) JM regions were substituted for the homologous domain in PTR. The former set of chimeras were active in PC12 cells whereas the latter were not, suggesting that the DDR1 KD lacks sufficient activity to produce differentiation in PC12 cells, even in the context of an otherwise activatable receptor structure.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Dr. J. Johnson (University of California, San
Francisco) kindly provided DDR1b cDNA. The antibodies, anti-Sos, PY20,
anti-Shc, and anti-PLC used for immunoprecipitation and Western blot
analyses were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz,
Calif.). Antibodies directed against the extracellular domain of PDGFR
and used for immunoprecipitation and Western blot analysis were
purchased from Genzyme (Cambridge, Mass.) and Austral (San Ramon,
Calif.), respectively. Dr. Joseph Schlessinger, New York
University, generously provided antisera against fibroblast growth
factor receptor substrate 2 (FRS2).
Chimeric receptor subcloning strategy
Receptor chimeras generally were prepared as described
previously (20
, 21)
. The 5' EcoRI/3'
MseI cDNA fragment of hPDGFRß encoding the amino-terminal
ED was ligated with a 5' MseI site introduced by polymerase
chain reaction (PCR) at the ED/TM junction of the cDNA encoding TrkA or
DDR1 TM/ID; it was flanked by a 3' EcoRI site. A
HindIII site in DDR1 located in the sequence encoding the
KD/JM junction, delineated by the G-X-G-X-X-G amino acid motif, was
ligated with a HindIII site introduced into the
corresponding sequence of TrkA by site-directed mutagenesis to generate
the coding sequence of the chimeras PDTR, PDTRb and their derivatives.
All the DNA sequences derived from PCR (including junction sites) were
sequenced and subcloned into the EcoRI site of pLEN for
stable transfection into PC12 cells. Expression of these constructs is
driven by LTR promoters.
Cell culture and stable transfection
The viral packaging cell line GP+e86 was transfected with the
pLEN retroviral chimera construct by the calcium phosphate method
described elsewhere (22)
. After 2 days, media from
transfected GP+e86 cells containing viral supernatant was filtered
(0.45 µM) and added to PC12 cells in the presence of polybrene. After
24 h, PC12 cells were subjected to G418 selection until individual
colonies could be selected and screened for expression of chimera by
Western blot analysis. Stably transfected PC12 cells were grown in
Dulbecco’s modified Eagles medium (DMEM) containing 2% plasma-derived
fetal calf serum, 5% plasma-derived horse serum, and 1%
penicillin/streptomycin.
PC12 differentiation assay
PC12 cells stably transfected with the chimera were seeded at a
density of 1 x 105 cells per well on
collagen coated 6-well tissue culture dishes in DMEM containing 1%
plasma-derived horse serum and 1% penicillin/streptomycin. Cells were
stimulated with 30 ng/ml PDGF or with media alone. The protein kinase C
(PKC) inhibitor bisindolymaleimide I (Calbiochem, La Jolla, Calif.) was
added 1 h prior to stimulation with growth factor at a
concentration of 1.0 µM. The kinetics and extent of growth
factor-induced neurite outgrowth were measured as the percentage of
cells with neurites longer than two cell bodies at specific times.
Photographs of the cells were taken and several fields counted (~200
cells).
Cell lysis and immunoprecipitation
After stimulation with 30 ng/ml PDGF for 10 min, culture media
were removed and the cells were washed in cold phosphate-buffered
saline containing 1 mM
Na3VO4. Lysis buffer (10 mM
Tris HCl pH 7.5, 5 mM EDTA, 1% Triton X-100, 50 mM NaCl, 30 mM sodium
pyrophosphate, sodium fluoride, 1 mM PMSF, 100 µM
Na3VO4) was added and cells
were lysed for 10 min on ice. Cell debris was removed by
centrifugation. Supernatants were transferred and the protein
quantitated. Antibodies (2–3 µg) were added to protein lysates and
the samples were mixed by rocking for 1–1/2 h at 4°C prior to
addition of protein A-Sepharose (Pharmacia, Piscataway, N.J.) for an
additional 1–1/2 h. The precipitates were washed three times in 1x
lysis buffer containing 100 µM
Na3VO4 and samples were
subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) before electroblotting to a PVDF membrane.
Immunoblot analysis
PVDF membranes were blocked at room temperature in Tris-buffered
saline (TBS) containing 3% bovine serum albumin (BSA) for 4 h.
Primary antibodies were diluted in TBS containing 3% BSA and incubated
with the membranes for 2 h at room temperature. After three washes
in TBS, the membranes were incubated for 1 h with the appropriate
horseradish peroxidase-conjugated secondary antibody (Jackson
ImmunoResearch Lab Inc. (New York, N.Y.) or Amersham (Arlington
Heights, Ill.) diluted in TBS containing 5% non-fat dry milk. After
three washes in TBS, the bands were visualized by ECL chemiluminesent
detection system (Amersham). Before reprobing, the membranes were
stripped in 0.2 M glycine-HCl pH 2.5, 0.05% Tween-20 at 80°C for
2 h.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 6)
.
To better evaluate the capacity of this receptor to signal in this
paradigm, chimeric receptors encoding the ED of hPDGFRß fused to the
TM/ID of DDR1 or DDR1b were stably transfected into PC12 cells, as has
been described for other RTKs (19
, 20)
. Despite high
levels of expression, particularly as compared to the positive control,
PTR (Fig. 2
B), the resulting chimeras (denoted PDR and PDRb) were unable
to mediate PC12 cell differentiation (data not shown) and displayed
undetectable levels of ligand-dependent receptor tyrosine
phosphorylation in response to short- (Fig. 2A
) and
long-term (data not shown) stimulation with PDGF. Consistent with these
findings, only transient and variably activation of ERK 1/2 was
observed in four different stably transfected clones, often at levels
too low to detect (data not shown).
|
As a result of the poor responses of the PDR chimeras in PC12 cells, a
new set of chimeras, in which the DDR KD was substituted by the TrkA
KD, were constructed. These ‘trimeras’ retained the advantages of the
PDR group with respect to PDGF regulation while affording the
opportunity to evaluate the signaling capacity/potential of the unusual
JM domains of DDR1 and DDR1b. The KD of TrkA provides the strong
catalytic activity that DDR1 KD appears to lack. Figure 3
shows the series of chimeras (and trimeras) constructed and their
salient features. The different ED, TM/JM, KD, and carboxyl-terminal
regions are represented as bars and the relevant tyrosine residues are
indicated. PTR, which has been shown to mediate differentiation of PC12
cells (19)
, was used as a positive control. In this
chimera (as in the native receptor), Y490 located in the JM region of
TrkA is required for Shc binding, and Y785 located in the carboxyl
terminus is necessary for PLC binding and activation. Both sites may
also contribute to binding of other signaling molecules such as Shp and
FRS2 (21
, 23)
. Mutagenesis of these tyrosine residues (to
phenylalanine) essentially eliminates the ability of TrkA (or PTR) to
bind and activate Shc and PLC, respectively. The PDR and PDRb
chimeras described above provide a negative control. The trimeras (PDTR
and PDTRb) contain the TM/JM domains of DDR1 or DDR1b and the KD and
carboxyl-terminal elements of TrkA. Thus, they lack the Y490 Shc
binding site of TrkA (or PTR); however, the longer JM of DDR1b encodes
a consensus Shc PTB binding site (N-P-A-Y) at Y513 (in DDR1b numbering)
(16)
.
|
Stably transfected PC12 cell lines expressing the various constructs
were plated onto 6-well collagen-coated dishes and scored for the
formation of neurites greater than two cell bodies in length at 24, 48,
and 72 h after the addition of PDGF (30 ng/ml) (Table 1
). The PTR chimera, as previously reported (19)
, completely
differentiates PC12 cells after 72 h exposure to ligand; this
response is not affected by the conversion of Y785 to phenylalanine,
which eliminates PLC binding (21)
. In contrast to PDR-
and PDRb-bearing cells, the PDTR- and PDTRb expressing cells
differentiate in response to ligand in a manner indistinguishable from
the PTR control. This suggests that the JM sequences of DDR1 and DDR1b
have signaling elements that allow for the recruitment and activation
of pathways that permit ligand-induced PC12 cell differentiation. In
contrast, a trimera composed of the ED of PDGFR, the TM/JM of TrkA, and
the KD and carboxyl terminus of DDR1 (PTDR) did not differentiate PC12
cells (data not shown). These results suggest that DDR1 contains a
kinase that is intrinsically weak or cannot be activated effectively in
this context. A comparable situation has been observed with FGFR4
relative to FGFR1, where the KD of the former is not as strongly
activated as the latter (albeit that it does signal in PC12 cells)
(24)
.
|
Since these chimeras (PDTR and PDTRb) also retain the PLC site of
TrkA, the contribution of this signaling entity to their responses was
assessed by converting the Y785 site located in the TrkA
carboxyl-terminal region to phenylalanine. The capacity of these
constructs (PDTR 785F and PDTRb 785F) to stimulate neurite formation
was substantially reduced, peaking at 25% and 35%, respectively
(Table 1)
; in the absence of additional ligand, the neurites were
unstable (data not shown). Thus, in contrast to PTR, the contribution
of PLC in the response of the trimeras is a major component in the
differentiation of these cells. This also suggests that the signals
generated through the JM region of DDR1 (or DDR1b) are either
qualitatively or quantitatively different from those arising from TrkA.
Although no evidence was obtained to suggest any qualitative
differences, this cannot be ruled out.
Stimulation of the PTR and PDTR chimeras with PDGF for 10 min produces
similar but distinct phosphorylation patterns of intracellular
signaling proteins (Fig. 4A
). Thus, the MAPKs ERK1/ERK2 are strongly activated in all
three chimeras, but the response is somewhat reduced by the
carboxyl-terminal Y785F mutation (and is essentially eliminated in
PDTRb 785F) (Fig. 4C
). This last observation is consistent
with the greatly reduced ability of PDTRb 785F to induce neuronal
differentiation (Table 1)
. In these experiments, receptor expression of
both PTR and the DDR1/TrkA chimeras were at comparable levels (Fig. 4B
).
|
Further dissection of the molecular signaling responses of these
chimeras is shown in Fig. 5
. Recruitment and activation of Sos, a guanine nucleotide exchange
factor, to the receptor complex is a key step in the activation of the
Ras/MAPK pathway, and adapter proteins such as Shc and FRS2 are
required to link receptors to Sos, usually via interactions with Grb2.
After stimulation of the indicated individual chimera expressing PC12
cell lines with PDGF, Sos was immunoprecipitated from cellular lysates
and the resulting Western blots were probed for SOS (Fig. 5A
) and for the association and tyrosine phosphorylation of
Shc and FRS2 (Fig. 5B, C
). Stimulation of cells expressing
PTR and PTR 785F chimeras, as expected, cause strong tyrosine
phosphorylation of both FRS2 and Shc that results in their association
with Sos (as compared to PC12 cell controls). PDTR and PDTR 785F are
also able to stimulate FRS2 tyrosine phosphorylation (Fig. 5B
) and subsequent association with Sos but, in contrast to
PTR, the involvement of Shc is greatly reduced (Fig. 5C)
. PDTRb and
PDTRb 785F, on the other hand, very weakly activate FRS2 (Fig. 5B)
, if
at all, but do tyrosine phosphorylate Shc, albeit not as strongly as
PTR (Fig. 5C
). Thus, the PDTRb chimera relies primarily on
Shc to recruit Sos and apparently lacks the FRS2 site found in PDTR.
|
Use of Shc by PDTRb to induce neuronal differentiation in this paradigm
is further emphasized by the results shown in Fig. 6
. After stimulation of native PC12 cells and PC12 cells expressing the
various chimeras, cell lysates were immunoprecipitated with Shc
antibodies and analyzed for the presence of receptors that would
indicate Shc–receptor complexes. PDTRb and PDTRb 785F, but not PDTR
and PDTR 785F, associate strongly with tyrosine phosphorylated Shc
(Fig. 6C, D
). PTR and PTR 785F do complex with Shc, but more
weakly than PDTRb, and are detected only after longer exposure times
(data not shown).
|
All the chimeras used in this study retain the PLC activation site
found in the carboxyl terminus of TrkA (Y785). The binding of PLC to
receptor tyrosine kinases is a necessary step in its tyrosine
phosphorylation and the stimulation of phospholipase activity. After
the addition of PDGF, lysates from the various cell lines were
immunoprecipitated with PDGFR antibody and analyzed for their
association with PLC (Fig. 7
). As expected, activated PTR, PDTR, and PDTRb associate with PLC;
PTR 785F, PDTR 785F, and PDTRb 785F do not (Fig. 7C
). As
indicated in Fig. 7A, B
, the receptors were expressed and
phosphorylated at comparable levels. These results indicate that
neither JM region of DDR1 contains a second PLC site and confirms
that the decrease in activity observed for the Y785F mutants of PDTR
and PDTRb is most likely due to loss of PLC activity.
|
Additional support for this view is provided by the PKC inhibitor
bisindoylmaleimide. PLC produces diacylglycerol that activates PKC,
which has been shown to phosphorylate ERKs (25)
. After
preincubation with bisindoylmaleimide and PDGF stimulation, PC12 cells
were assayed for their ability to form neurites as described above
(Table 2
). The PKC inhibitor had little effect on PTR-mediated neurite
formation, consistent with the view that PLC is unnecessary for
NGF-induced neurite outgrowth; however, marked inhibition of
differentiation was apparent for PDTR and PDTRb, supporting the
conclusion that PLC is a necessary part of the trimera response.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the case of RTKs, the mature receptor functions as a dimer in which
the two protomers are held in an orientation that allows activation of
the kinase by the ligand (1)
. It is unclear, in all cases,
whether the ligand induces the dimerization or whether it binds to
preformed dimers and, in so doing, changes the protomer orientations
(26)
. In either case, the kinase activation likely results
from the shift of the ‘lip’ segment (that contains the site(s) of
initial autophosphorylation) from an inhibitory conformation (i.e.,
sterically blocking the catalytic site) to one that allows free
enzyme–substrate interaction (27
, 28)
. The subsequent
phosphorylation presumably provides additional interactions with other
sites on the protein, probably of an electrostatic nature, to stabilize
the open (active) conformation.
The DDRs, both by the nature of the identified ligands and in the
responses induced, are distinctly different. Their reaction to collagen
binding, the only identified ligand to date, is neither rapid nor
transitory and, at least, in the cell model tested (human 293 cells),
produced no detectable downstream signaling or effects
(5
6
7)
. The reasons for this are unclear and could be due
to many things, ranging from the technical to the physiological. Thus,
the right models to test DDR responses may not have been formulated
yet; when proper conditions are found, including other ligands (or
larger complexes of the presently known ligands), these receptors may
be shown to behave more like other RTKs. However, it is also possible
that the DDRs represent a fundamentally new class of receptors whose
signaling mechanisms are substantially different from the presently
known members of the superfamily of RTKs.
As one approach to determining the bases for the very low levels of
detectable enzymatic activity in DDR isoforms, the extracellular domain
was replaced with that of PDGFR and the constructs (PDR) stably
transfected with PC12 cells. Similar derivatives with the TM/KDs of
TrkA (19)
, FGFR1, 3, and 4 (20
, 24)
and EGF
(S. Layden and R. A. Bradshaw, unpublished data) are highly active
in this milieu and rapidly induce neurite proliferation (as well as
various underlying molecular signaling events) in a ligand-dependent
fashion. The PDGFR EDs have been shown to form pre-dimers
(29)
; in one case, a mutant of FGFR3, the pre-dimer
neutralizes the ligand-independent activation of the kinase (in the
protomeric state) (28)
. However, even with the PDGFR ED
interactions, neither chimera (PDR or PDRb) induced PC12
differentiation or showed significant autophosphorylation. The
constructs effectively eliminate inability to form dimers as a cause of
low activity and provide further support for the view that the low
levels of activity result from characteristics of either the JM or KDs,
or both.
To further ascertain the relative contributions of each of these
domains, the constructs were prepared with the corresponding domains of
TrkA interchanged. When tested as stable transfectants, the resulting
trimeras clearly indicated that the DDR1 KD was unable to use the TrkA
JM (which contains Y490, a site that produces full differentiation in
native TrkA). This same domain (TrkA JM) in the context of the EGFR
also induces ligand-dependent differentiation (30)
. The
TrkA KD could use either DDR1 JM, except that these trimeras
differentially used Shc and FRS2 and, unlike TrkA, were unable to exert
full activity without PLC activation (provided by the Y785 site of
the TrkA carboxyl-terminal). The principal interactions of the various
chimeric receptors studied with the germane signaling entities
identified in these studies are summarized in Fig. 8
.
|
It is unclear why the activation of FRS2 and Shc by PDTR and PDTRb,
respectively, is insufficient to induce full differentiation of PC12
cells. FRS2 presumably binds through its PTB site (located at the
NH2 terminus of that molecule), but it may or may
not involve a phosphotyrosine in the DDR1 JM. Similar interactions with
the JM of the FGFR1 do not involve phosphotyrosine (29
;
E. D. Foehr and R. A. Bradshaw, unpublished results). As
judged by Sos immunoprecipitation (Fig. 5)
, which presumably measures
the amount of activated FRS2, neither PDTR nor PDTR 785F is as
effective as PTR (or PTR 785F) in activating FRS2. A similar
observation was made for Shc activation by PDTRb. However, in that
case, direct immunoprecipitation with anti-Shc antibodies showed a
substantially greater association of Shc with PDTRb (or the 785F
derivative). Under the same conditions, PTR bound much less Shc (in
fact, longer exposures are necessary to generate a strong signal).
However, the amounts of activated Shc, as judged by phosphorylation in
each precipitate (PTR vs. PDTRb), were comparable. These results
suggest that Shc binding to the PDTRb JM is strong, but that the
resulting phosphorylation (activation) is much less than that observed
for PTR and that possibly some of the Shc is not in a form useful for
Sos interaction and Ras activation. Shc has been shown to be
phosphorylated on both Y317 and Y239/Y240 (31
32
33)
, but
only the Y239/Y240 site is functional in activating Ras in PC12 cells
(by TrkA) (33)
. Thus, the PDTRb JM site may be
functionally weaker even though the amount of complex formed is
substantial (potentially much greater than with PTR).
Table 3
summarizes the various responses for each of the chimeras studied. It
appears that the activation of any two of these three pathways (Shc,
FRS2, and PLC) can produce full differentiation. However, it should
be emphasized that the level of each response is likely to be as
important as which combination of signals is produced. In PTR 785F,
where the PLC contribution is eliminated, both Shc and FRS2 can be
(and are) activated. However, these interactions are substantially
reduced by converting Y490 to phenylalanine (Shc is virtually
eliminated), and thus both may compete for the same site, i.e., only
one of the two can be activated at any given time (E. D. Foehr, E.
Tanabe, A. Tatavos, and R. A. Bradshaw, unpublished results).
Whether either one alone would be sufficient to give full
differentiation in NGF-stimulated cells is unknown.
|
These results confirm that DDR JM regions can be activated and are capable of binding and using at least two signaling molecules, although not demonstrably by DDR1 KDs. It remains to be established whether these sites are used in DDR1 (or DDR1b) in a germane physiological context.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
|---|
2 Present address: Rehsteig 8, Leuting, Germany.
3 Present address: Laboratory of Tissue Engineering, I.D.I. Istituto Dermopatico dell’Immacolata, Via dei Castelli Romani, Rome, Italy.
Received for publication October 7, 1999. Revised for publication December 21, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 W. Matsuyama, M. Faure, and T. Yoshimura Activation of Discoidin Domain Receptor 1 Facilitates the Maturation of Human Monocyte-Derived Dendritic Cells Through the TNF Receptor Associated Factor 6/TGF-{beta}-Activated Protein Kinase 1 Binding Protein 1{beta}/p38{alpha} Mitogen-Activated Protein Kinase Signaling Cascade J. Immunol., October 1, 2003; 171(7): 3520 - 3532. [Abstract] [Full Text] [PDF]
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 C. A. Curat, M. Eck, X. Dervillez, and W. F. Vogel Mapping of Epitopes in Discoidin Domain Receptor 1 Critical for Collagen Binding J. Biol. Chem., November 30, 2001; 276(49): 45952 - 45958. [Abstract] [Full Text] [PDF]
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 W. F. Vogel, A. Aszódi, F. Alves, and T. Pawson Discoidin Domain Receptor 1 Tyrosine Kinase Has an Essential Role in Mammary Gland Development Mol. Cell. Biol., April 15, 2001; 21(8): 2906 - 2917. [Abstract] [Full Text]
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 C. Grayson, S. N.M. Reid, J. A. Ellis, A. Rutherford, J. C. Sowden, J. R.W. Yates, D. B. Farber, and D. Trump Retinoschisin, the X-linked retinoschisis protein, is a secreted photoreceptor protein, and is expressed and released by Weri-Rb1 cells Hum. Mol. Genet., July 22, 2000; 9(12): 1873 - 1879. [Abstract] [Full Text] [PDF]
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E. D. Foehr, S. Raffioni, J. Murray-Rust, and R. A. Bradshaw The Role of Tyrosine Residues in Fibroblast Growth Factor Receptor 1 Signaling in PC12 Cells. SYSTEMATIC SITE-DIRECTED MUTAGENESIS IN THE ENDODOMAIN J. Biol. Chem., September 28, 2001; 276(40): 37529 - 37536. [Abstract] [Full Text] [PDF]
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PY) antibody and B) anti-PDGFRß (
