From the Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114
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ABSTRACT |
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Activation of receptor tyrosine kinases is
thought to involve ligand-induced dimerization, which promotes receptor
transphosphorylation and thereby increases the receptor's
phosphotransferase activity. We used two platelet-derived growth factor
-receptor (
-PDGFR) mutants to identify events that are required
for full engagement (autophosphorylation and activation of the kinase
activity) of the
-PDGFR kinase. The F79/81 receptor (Tyr to Phe
substitution at 579 and 581 in the juxtamembrane domain of the
receptor) was capable of only very modest ligand-dependent
autophosphorylation and also failed to associate with numerous SH2
domain-containing proteins. Furthermore, stimulation with
platelet-derived growth factor (PDGF) did not increase the kinase
activity of the F79/81 mutant toward exogenous substrates. However, the
F79/81 receptor had basal kinase activity and could be artificially
stimulated by incubation with ATP. Because the low kinase activity of
the F857 mutant (Tyr to Phe substitution at 857 in the putative
activation loop) could not be increased by incubation with ATP, failure
to phosphorylate Tyr-857 may be the reason why the F79/81 mutant has
low kinase activity. Surprisingly, the F857 mutant underwent efficient
PDGF-dependent autophosphorylation. Thus the
PDGF-dependent increase in the kinase activity of the
receptor is not required for autophosphorylation. We conclude that full
activation of the
-PDGFR kinase requires at least two, apparently
distinct events.
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INTRODUCTION |
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Receptor tyrosine kinases are a large family of
transmembrane-spanning proteins that transmit signals from the
extracellular environment by means of activating their intrinsic kinase
activity. The ligand for the platelet-derived growth factor
-receptor (
-PDGFR)1
exists as a dimer, and on binding to the receptor it induces dimerization (1). Much work has indicated that dimerization is both
necessary and sufficient to activate the kinase activity of the
receptor, leading to autophosphorylation of the receptor and the
subsequent binding and phosphorylation of downstream signaling proteins
(reviewed in Refs. 2-5), but the precise steps involved in the
mechanism of receptor activation remain unknown. Receptor activation is
the first step in the increasingly complex signaling cascades induced
by receptor tyrosine kinases; therefore a complete understanding of the
activation process is important.
Many tyrosines in the intracellular portion of the -PDGFR have been
shown to be phosphorylated in response to ligand binding to the
extracellular portion of the receptor. Phosphorylation of tyrosine 857 in the putative kinase activation loop has been shown to be required
for maximal receptor kinase activity (6, 7). Many other
phosphotyrosines act as docking sites for SH2 domain-containing
proteins such as the GTPase-activating protein of Ras (RasGAP) (8),
phospholipase C
-1 (PLC
) (9-12), and the p85 subunit of PI
3-kinase (p85) (13-16). PDGF-dependent association of Src family
members requires tyrosines 579 and 581 in the juxtamembrane region of
the human
-PDGFR (17, 19). Mutation of either tyrosine 579 or 581 individually led to a decrease in Src association with the receptor
(17), whereas mutation of both sites led to a receptor that failed to
detectably associate Src (19) and has been reported to be devoid of
ligand-stimulatable kinase activity (17).
We constructed and characterized a -PDGFR mutant with tyrosines 579 and 581 mutated to phenylalanine (referred to as F79/81) and confirmed
the finding that these sites are required for association of Src with
the receptor (17-19). In addition we found that the
-PDGFR did not
become efficiently tyrosine-phosphorylated in response to PDGF and as a
result was unable to recruit wild type levels of SH2 domain-containing
proteins. However, in contrast to previous findings (17) we found that
the F79/81 receptor was not devoid of kinase activity, with the basal
phosphorylation of the F79/81 receptor being comparable or slightly
higher than that of the WT receptor. In addition we found that the
F79/81 receptor kinase activity could be artificially activated by
incubation with ATP. To investigate the defect in the ligand-induced
activation of the F79/81 receptor we compared it to another
-PDGFR
mutant where tyrosine 857 was replaced with phenylalanine. In contrast to the F79/81 receptor, the F857 receptor became highly
tyrosine-phosphorylated following exposure to PDGF. However, despite
being highly tyrosine-phosphorylated the F857 receptor was not
activated as a kinase toward exogenous substrates. Comparison between
the F79/81 and F857 receptors indicated that there are at least two
distinct steps in receptor activation, autophosphorylation that
requires tyrosines 579 and 581 in the juxtamembrane region and
phosphorylation of tyrosine 857 that allows the receptor to
phosphorylate exogenous substrates.
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MATERIALS AND METHODS |
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Cell Lines--
HepG2 cells, a human hepatoma cell line that
does not express endogenous -PDGF receptors, were cultured and
maintained as described previously (20).
Construction and Expression of -PDGF Receptor
Mutants--
Substitutions of phenylalanine for tyrosine at positions
579 and 581 in the human
-PDGF receptor were performed by
site-directed mutagenesis as described previously (19). The mutated
receptor DNA was subcloned into the pLXSN2 vector and
introduced into the
2 and PA317 packaging cell lines, and the
resulting virus was used to infect parental HepG2 cells (20). The
infected cells were selected in the presence of 1 mg/ml (active
concentration) G418, and the resulting mass populations were sorted by
fluorescence-activated cell sorter using the human
-PDGF
receptor-specific monoclonal antibody PR7212 (Genzyme) to obtain cell
lines expressing comparable levels of receptors, previously estimated
to be 5 × 105 receptors/cell (20).
Immunoprecipitation and Western Blotting--
HepG2 cells were
grown to 80% confluence and then incubated for 16-20 h in Dulbecco's
modified Eagle's medium containing 0.1% fetal bovine serum. Cells
were then treated with 40 ng/ml PDGF-BB or with buffer alone at
37 °C for the indicated times. Cells were washed twice with HS (20 mM Hepes, pH 7.4, 150 mM NaCl) and then lysed
in EB (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 1% Triton X-100, 0.1%
BSA, 20 µg/ml aprotinin, 2 mM
Na3VO4, 1 mM phenylmethylsulfonyl
fluoride) (22). Lysates were centrifuged for 20 min at 12,000 × g, and receptors were immunoprecipitated from the soluble
fraction with the 30A antiserum, which is specific for the -PDGFR
(24). Immune complexes were bound to formalin-fixed membranes of
Staphylococcus aureus, spun through an EB sucrose gradient, and then washed twice with EB, PAN (10 mM Pipes,
pH 7.0, 100 mM NaCl, 1% aprotinin) + 0.5% Nonidet P-40,
and PAN (23) and resuspended in PAN before being used in kinase assays
or analyzed by Western blotting.
Kinase Assays--
-PDGFR immunoprecipitates were subjected
to a standard in vitro kinase assay (6) in the presence of
100 µM peptide (MAEEEEYVFIEAKKK) or 0.5 µg of
GST-PLC
and 10 µCi of
-[32P]ATP in universal
kinase buffer (UKB) (20 mM Pipes, pH 7.0, 10 mM
MnCl2, 20 µg/ml aprotinin). To pretreat samples with
unlabeled ATP, immunoprecipitates were incubated with UKB containing 10 µM ATP for 20 min at 30 °C and then washed once with
PAN before being subjected to an in vitro kinase assay.
Phosphorylated peptides were separated from
-[32P]ATP
on cellulose plates by thin layer electrophoresis at pH 3.5, and
phosphorylated GST-PLC
was separated by 7.5% SDS-PAGE. The level of
phosphorylation of peptide or GST-PLC
was quantitated using
Molecular Dynamics PhosphorImager IQ software.
Preparation of Pervanadate-- Equal volumes of 20 mM H2O2 and 2 mM Na3VO4 were combined at room temperature for 10 min. The resulting solution was added to intact cells to a final concentration of 1 mM H2O2 and 0.1 mM Na3VO4.
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RESULTS AND DISCUSSION |
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Tyrosines 579 and 581 in the juxtamembrane region of the human
-PDGFR were mutated to phenylalanine (19), and the resulting receptor (F79/81) was expressed in HepG2 cells. HepG2 cells express a
very low level of PDGF
-receptor and no detectable
-PDGFRs (29).
It had been previously reported that mutation of tyrosines 579 and 581 resulted in a kinase-inactive receptor (17) or a receptor with reduced
kinase activity (19). Therefore, we first compared the ability of the
F79/81 receptor to become phosphorylated in response to PDGF with the
WT receptor and a receptor with a mutation at tyrosine 857 (F857)
previously shown to be required for full kinase activity (6, 7). HepG2
cells expressing these receptors were exposed to 40 ng/ml PDGF-BB for 5 min and then lysed and
-PDGFR-immunoprecipitated. A portion of the
immunoprecipitates representing approximately 1.5 × 106 cells was analyzed for phosphotyrosine content and
receptor levels by Western blot (Fig. 1).
Fig. 1A shows that the WT receptor had undetectable levels
of phosphorylation in resting cells and became highly
tyrosine-phosphorylated after exposure to PDGF. Surprisingly the F857
receptor also became highly tyrosine-phosphorylated in response to
PDGF, although levels of phosphorylation were slightly reduced compared
with WT. Note that part of the apparently lower F857 phosphorylation
compared with WT following PDGF stimulation was because of there being
less F857 that WT receptor (Fig. 1B). In contrast to the WT
and F857 receptors, the F79/81 receptor was very poorly phosphorylated
following addition of PDGF despite approximately equal amounts of
receptor being present in the samples (Fig. 1B). Increasing
the length of time that the cells were exposed to PDGF had no effect on
the level of tyrosine phosphorylation of the F79/81 receptor (data not
shown).
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Phosphorylation of the -PDGFR is required for the association of
many SH2 domain-containing proteins. Our finding that the F79/81
receptor was poorly phosphorylated suggested that the Src family
members may not be the only SH2-containing proteins that cannot
associate efficiently with the F79/81 receptor. Consequently, we tested
the ability of the F79/81 receptor to associate with a number of
proteins that have been shown to bind to the WT receptor following PDGF
stimulation. Receptors were immunoprecipitated from resting or
stimulated cells and subjected to Western blot analysis for the
-PDGFR, PLC
, RasGAP, and the p85 subunit of PI 3-kinase (p85). As
shown in Fig. 2A, the F79/81
receptor bound reduced amounts of PLC
and p85 and undetectable
levels of RasGAP compared with the WT receptor. The F857 receptor bound
the proteins to levels similar as the WT receptor. The kinase dead
receptor (R634) included as a control did not detectably bind any of
the proteins that associate with the WT receptor.
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Like the receptor-associated proteins described above, Src family
members bind to the -PDGFR directly, via the SH2 domain (18, 42). We
and others have found that the best way to detect this interaction is
by immunoprecipitating Src and looking for associated receptor. Fig.
2B shows that the amount of receptor coprecipitating with
Src increased markedly in response to PDGF, whereas no receptor was
detected in Src immunoprecipitates from F79/81 or kinase dead
receptor-expressing cells, regardless of the presence of PDGF. The
amount of receptor-Src complex that was detected in PDGF-stimulated
cells expressing receptors singly mutated at either 579 or 581 was also
dramatically reduced. In contrast, mutating either of these tyrosines
individually had much less of an effect on receptor autophosphorylation
and recruitment of SH2 domain-containing proteins, although mutating
tyrosine 579 had more of an effect than substituting tyrosine 581 (data not shown).
The data presented in Fig. 2 are consistent with previous findings that
Src associates with the -PDGFR provided that tyrosines 579 and 581 are phosphorylated (17, 19). Our findings indicate that the F79/81
receptor is not a selective Src binding mutant since the binding of
other SH2 domain-containing proteins to the F79/81 receptor was also
compromised. This was not a surprising result because we and others
showed that the F79/81 receptor was very poorly tyrosine-phosphorylated
following ligand stimulation (Fig. 1) (17, 19). In agreement with
Vaillancourt et al. (19) we found that the amount of F79/81
receptor phosphorylation could be modestly increased following ligand
stimulation, whereas Mori et al. (17) found that this
receptor was not detectably phosphorylated. The difference between our
data and that of Mori et al. (17) could be related to the
cell type used, as we expressed the receptors in HepG2 cells whereas
Mori et al. (17) used pig aortic endothelium cells. When
expressed in other cell types (PC12 (19) or Ba/F3 cells (25)) the
F79/81 receptor did show some receptor autophosphorylation and kinase
activity, similar to the data presented here for HepG2 cells. Because
none of the cell lines used for these studies naturally express the
-PDGFR, it is possible that they do not accurately reflect the
behavior of the F79/81 receptor in a natural setting. We have expressed
a chimeric
/
F72/74 receptor (26) in Ph cells, which are a
3T3-like cell line that does not express the
-PDGFR, as well as in
HepG2 cells. This chimeric receptor mutant behaved comparably when
expressed in these two cell
lines.2 These findings
indirectly indicate that the behavior of the F79/81 receptor is not
dramatically altered by expressing it in the HepG2 cells. Ongoing
studies, in which the mutant receptors will be expressed in mesenchymal
cells devoid of either
- or
-PDGFRs, will better address these
issues.
One explanation for the failure of PDGF to promote the phosphorylation of the F79/81 receptor is that the mutant receptors are unable to dimerize correctly. This does not appear to be the case because the ligand binding domain is within the first three IgG loops in the extracellular domain, and mutations within the cytoplasmic domain have not been shown to inhibit ligand binding or dimerization of the receptors. Furthermore, mutation of tyrosine 579 or 581 alone had no effect on ligand binding (17). Finally, we found that the F79/81 and WT receptors underwent a comparable extent of the PDGF-dependent dimerization (data not shown). These data indicate that replacing tyrosines 579 and 581 with phenylalanine did not prevent binding of PDGF to the extracellular domain of the receptor or receptor dimerization.
A second possibility is that the phosphorylation of the juxtamembrane sites makes a very large contribution to the overall phosphorylation of the receptor. However, it has been shown that in vivo the most highly phosphorylated sites in the receptor are at tyrosines 857 and 751 (16). In addition, the phosphorylation of sites 579 and 581 was not detectable by standard phosphopeptide mapping techniques suggesting that they are phosphorylated to low stoichiometry in vivo (17). Mutation of tyrosine 751 alone or in combination with up to four other tyrosines involved in binding-associated proteins did not prevent the receptor from undergoing extensive tyrosine phosphorylation in vivo (27). Therefore it is unlikely that the decrease in phosphorylation of the F79/81 receptor is because of the lack of two phosphorylation sites.
Because the activation of receptor tyrosine kinases has been
inextricably linked with receptor phosphorylation, we next tested the
kinase activity of the -PDGFR mutants. WT, F857, and F79/81 receptors were immunoprecipitated from resting or PDGF-BB-stimulated cells, and immunoprecipitates representing approximately 3 × 105 cells were subjected to an in vitro kinase
assay in the presence of a peptide substrate with a tyrosine residue in
a sequence shown to be the optimal phosphorylation site for the
-PDGFR (28). Peptides were separated from the
-[32P]ATP on cellulose plates by thin layer
electrophoresis, and the degree of phosphorylation of the peptides was
quantitated using a PhosphorImager. Fig.
3A shows that the WT receptor
immunoprecipitated from PDGF-treated cells phosphorylates the peptide
approximately four times better than receptors from resting cells.
However, PDGF treatment of cells expressing the F79/81 or F857
receptors did not detectably increase the kinase activity of either
receptor toward the peptide substrate. We altered the time of the assay incubation (Fig. 3C) and the substrate concentration (Fig.
3D), but neither of these variables improved the activity of
the F79/81 or F857 receptors. In addition, increasing the time of
exposure of the cells to PDGF prior to precipitation of the receptors
did not result in activation of either the F79/81 or the F857 receptors (data not shown). We also tested the activity of the receptor mutants
against a GST-PLC
fusion protein substrate (29) and found WT
receptor phosphorylated this substrate approximately six times better
following PDGF stimulation. Addition of PDGF to cells expressing the
F79/81 and F857 receptors had very little effect on the ability of
either receptor to phosphorylate GST-PLC
(Fig. 3B).
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Whereas the kinase activity of the F79/81 and F857 receptors was not increased in response to PDGF, these receptors did show basal tyrosine phosphorylation that was greater than that of the WT receptor (Fig. 1) and basal kinase activity that was comparable with the WT (data not shown). Because the receptors were not catalytically dead we tested whether they could be artificially activated. WT, F79/81, and F857 receptors were immunoprecipitated from resting cells and incubated with 10 µM ATP prior to being assayed for activity against the peptide substrate. Fig. 4 shows that incubation of F79/81 receptor with ATP caused the receptor to become activated to the same extent as the WT receptor. In contrast, the F857 receptor was poorly activated by this treatment. These data indicate that the F79/81 receptor is capable of being artificially activated. Furthermore, the failure of the F857 receptor to be activated by incubation with ATP suggests that the F79/81 receptor kinase is not activated in response to PDGF in vivo because it does not become phosphorylated at tyrosine 857.
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A method to increase the phosphotyrosine content of proteins in
vivo is to treat cells with the nonspecific tyrosine phosphatase inhibitor sodium pervanadate (30). We reasoned that this could be a way
to phosphorylate and to potentially activate the F79/81 and F857
receptors in vivo. Cells were treated with sodium
pervanadate for 2 min before being lysed, and then the -PDGFRs were
immunoprecipitated, and a portion of the immunoprecipitate representing
1.5 × 106 cells was analyzed for phosphotyrosine
content by Western blotting and the blots stripped and reprobed for
-PDGFR. Fig. 5 shows that the WT,
F79/81, and F857 receptors all became highly phosphorylated in sodium
pervanadate-treated cells (Fig. 5A). The apparently lower
extent of phosphorylation of the F857 receptor with all treatments
(Fig. 5A) was because of a reduced amount of receptor in the
immunoprecipitates (Fig. 5B). Thus pervanadate treatment of
cells was able to stimulate robust tyrosine phosphorylation of all the
receptors. We next tested the receptors immunoprecipitated from sodium
pervanadate-treated cells for kinase activity toward the peptide
substrate. Immunoprecipitates representing 3 × 105
cells were assayed for activity against the peptide substrate. Fig.
5C shows that treatment of the cells with sodium pervanadate failed to increase the kinase activity of any of the receptors toward
the peptide substrate. These data show that sodium pervanadate treatment of cells does not increase the kinase activity of the receptors toward exogenous substrates, despite extensive tyrosine phosphorylation of the receptors. Given that the phosphorylation of
tyrosine 857 appears to be required for activating the receptor's kinase activity toward exogenous substrates it is possible that this
tyrosine is inaccessible for phosphorylation when receptors are
monomeric.
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If receptor dimerization was a prerequisite for phosphorylation of
Tyr-857, then treating cells with pervanadate and PDGF should activate
the F79/81 receptor. To test this possibility, cells were treated for 2 min with sodium pervanadate, and then the medium was removed and
replaced with fresh medium containing PDGF for 5 min before cells were
lysed and receptors immunoprecipitated. Fig. 5A shows that
this treatment led to highly phosphorylated receptors, in the same way
as sodium pervanadate treatment alone. This treatment also increased
the kinase activity of the WT receptor to the same extent as PDGF alone
(Fig. 5C), indicating that the phosphorylation of the
receptor because of sodium pervanadate treatment does not prevent or
enhance activation of the WT receptor kinase following ligand binding.
However, pretreatment with sodium pervanadate followed by the addition
of PDGF did not give any increase in the kinase activity of the F79/81
or F857 receptors. Fig. 5C also shows that the
kinase-inactive form of the -PDGFR (R634) became highly
tyrosine-phosphorylated following sodium pervanadate treatment. This
indicates that a kinase other than the receptor itself is responsible
for the phosphorylation seen following sodium pervanadate treatment. In
summary, the lack of activation of the F79/81 receptor following
pervanadate and PDGF treatment could be because the sites that need to
be phosphorylated to achieve an activated kinase are not phosphorylated
in the presence of sodium pervanadate.
Because the levels of receptor phosphorylation and kinase activity
are so profoundly affected by mutation of tyrosines involved in
binding Src, it is possible that the defect in the F79/81 is because of
a lack of Src association with the -PDGFR. Previous studies have
shown that Src will phosphorylate and activate the receptors for
insulin and insulin-like growth factor 1 in vitro (31-33),
and Src has been shown to phosphorylate tyrosine 934 of the
-PDGFR
both in vitro and in vivo (34). In addition,
receptor tyrosine kinases appear to be activated when c-Src is
overexpressed or when activated forms of Src are expressed (35-37).
However, under a variety of in vitro conditions we could not
show
-PDGFR receptor tyrosine phosphorylation by
Src.3 Although this does not
rule out a role for Src acting through intermediates or the possibility
that Src phosphorylates the receptor in vivo, our data
suggest that the role of tyrosines 579 and 581 in PDGF receptor
activation is not a direct effect of receptor phosphorylation by
Src.
Several groups have shown that phosphorylation at tyrosine 857 is
required for full activation of the -PDGF receptor (6, 7). Based on
the crystal structure of the receptors for insulin and fibroblast
growth factor, kinase activity is suppressed by an "activation
loop," which sterically blocks the catalytic site (38, 39).
Phosphorylation of one or more tyrosine residues in the activation loop
removes the steric hindrance and allows substrates access to the
catalytic site. More recent crystals of Src family members have
suggested that the activation of the kinase involves movement of
-helix C, which leads to conformational changes that lead to
increased kinase activity (40, 41). The phosphorylation of the
activation loop may also be involved in the movement of
-helix C. The crystal structure of the
-PDGFR is yet to be solved, but based
on sequence comparisons tyrosine 857 is located in a region homologous
to the activation loop seen in other receptors, and a role for this
site in receptor activation can be postulated. Consistent with tyrosine
857 being involved in repression of
-PDGFR kinase activity, when the
site is mutated to phenylalanine (F857), the basal level of receptor
phosphorylation is higher than that see in the wild type receptor (Fig.
1A).
In summary we have shown that the F79/81 receptor is not selectively impaired in binding Src but that it is also deficient in associating with a number of other SH2 domain-containing proteins. The basis for the defect in association with SH2 domain-containing proteins appears to be that the F79/81 receptor fails to become efficiently phosphorylated in response to the binding of PDGF. In addition to the defect in binding-associated proteins, the F79/81 receptor kinase activity was not activated toward exogenous substrates in response to PDGF stimulation. Comparison of the tyrosine phosphorylation and kinase activity of the F79/81 and F857 receptor mutants in response to ligand stimulation leads us to propose a two-step model for the full activation of the PDGF receptor. This model proposes that the two juxtamembrane tyrosines at 579 and 581 are required for receptor autophosphorylation, which is a prerequisite for the later phosphorylation of tyrosine 857, which is necessary for the activation of the receptor kinase activity.
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ACKNOWLEDGEMENTS |
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We thank members of the Kazlauskas laboratory for critically reading this manuscript. We thank Arlen Thomas (Amgen) for the gift of PDGF-BB.
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FOOTNOTES |
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* This research was supported by Grant EY 11693 from the National Institutes of Health (to A. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Cutaneous Biology Research Center, Massachusetts
General Hospital, 149 13th St., Charlestown, MA 02129.
§ Present address: Cadus Pharmaceutical, 777 Old Saw Mill River Rd., Tarrytown, NY 10591.
¶ Present address: Dept. of Pharmacology and Toxicology, University of Arizona College of Pharmacy, Tuscon, AZ 85721.
Established Investigator of the American Heart Association. To
whom correspondence should be addressed: Schepens Eye Research Inst.,
Harvard Medical School, 20 Staniford St., Boston, MA 02114. Tel.:
617-912-2517; Fax: 617-912-0111; E-mail:
kazlauskas{at}vision.eri.harvard.edu.
1
The abbreviations used are: -PDGFR,
platelet-derived growth factor
-receptor; PDGF, platelet-derived
growth factor; F79/81, tyrosines 579 and 581 mutated to phenylalanine;
F857, tyrosine 857 mutated to phenylalanine. PLC, phospholipase C; PI,
phosphatidylinositol; WT, wild type; BSA, bovine serum albumin; Pipes,
1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel
electrophoresis; GST, glutathione S-transferase; R634,
kinase-inactive receptor where lysine at 634 is mutated to
arginine.
2 K. DeMali and A. Kazlauskas, unpublished observations.
3 R. M. Baxter, J. P. Secrist, R. R. Vaillancourt, and A. Kazlauskas, unpublished observations.
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REFERENCES |
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