(Received for publication, February 21, 1996)
From the
Screening of a human breast epithelial cell cDNA library with
the tyrosine-phosphorylated C terminus of the epidermal growth factor
receptor identified a novel member of the GRB7 gene family,
designated GRB14. In addition to a pleckstrin homology
domain-containing central region homologous to the Caenorhabditis
elegans protein F10E9.6/mig 10 and a C-terminal Src homology 2
(SH2) domain, a conserved N-terminal motif, P(S/A)IPNPFPEL, can now be
included as a hallmark of this family. GRB14 mRNA was
expressed at high levels in the liver, kidney, pancreas, testis, ovary,
heart, and skeletal muscle. Anti-Grb14 antibodies recognized a protein
of approximately 58 kDa in a restricted range of human cell lines.
Among those of breast cancer origin, GRB14 expression strongly
correlated with estrogen receptor positivity, and differential
expression was also observed among human prostate cancer cell lines. A
GST-Grb14 SH2 domain fusion protein exhibited strong binding to
activated platelet-derived growth factor (PDGF) receptors (PDGFRs) in vitro, but association between Grb14 and -PDGFRs could
not be detected in vivo. In serum-starved cells, Grb14 was
phosphorylated on serine residues, which increased with PDGF, but not
EGF, treatment. Grb14 is therefore a target for a PDGF-regulated serine
kinase, an interaction that does not require PDGFR-Grb14 association.
Many intracellular targets for receptor tyrosine kinases (RTKs) ()contain one or more SH2 domains. These are conserved,
noncatalytic domains of approximately 100 amino acids that bind to
short peptide sequences containing phosphotyrosine (1) . Since
receptor autophosphorylation on specific tyrosine residues follows RTK
activation, SH2 domains mediate receptor-substrate interactions as well
as other protein-protein interactions during signal transduction. Since
the specificity of SH2 domain binding is largely determined by amino
acid residues C-terminal to the phosphotyrosine, the particular
autophosphorylation sites present on a given RTK define the SH2
domain-containing signaling proteins that it can recruit and hence, to
a large extent, the signaling specificity of the receptor. The CORT
technique, in which cDNA expression libraries are screened with the
tyrosine-phosphorylated C terminus of the EGFR, represents a powerful
methodology for the identification and characterization of novel, SH2
domain-containing, receptor substrates (2, 3, 4, 5) .
SH2 domains are
often accompanied in signaling proteins by two other conserved protein
modules: SH3 domains, which bind to proline-rich peptide ligands with a
PXXP core sequence (6) and thereby also mediate
protein-protein interactions, and PH domains. The latter are conserved
protein modules now identified in about 60 intracellular proteins, most
of which either perform a signaling function or are associated with the
membrane cytoskeleton(7) . Despite the frequent occurrence of
the PH domain and the recent definition of its three-dimensional
structure (8, 9, 10, 11) the precise
role of this module remains obscure. Although several PH domains bind
to the subunits of heterotrimeric G proteins(12) ,
this interaction appears to involve only the C-terminal region of the
domain. Two other groups have reported protein-protein interactions
mediated by PH domains; Yao et al. observed binding of the Btk
PH domain to protein kinase C(13) , whilst the PH domain of Akt
contributes to homotypic oligomerization and kinase
regulation(14) . However, another possibility is that not all
PH domain ligands are protein in nature, since several of these domains
bind phosphatidylinositol 4,5-bisphosphate(15) , and inositol
1,4,5-trisphosphate represents a high affinity ligand for the
phospholipase C-
PH domain (16) .
SH2
domain-containing proteins can be divided into two
classes(17) : class I, which also possess a catalytic function, e.g. phospholipase C-1 and Ras-GAP, and class II, which
contain only noncatalytic protein modules and are thought to function
as adaptors, linking separate catalytic subunits to receptors or other
signaling proteins. A member of the latter class is Grb2, which
consists of a SH2 domain flanked by two SH3 domains. The SH2 domain
acts as a binding site for specific tyrosine-phosphorylated proteins
including the EGFR and Shc, while the SH3 domains bind proline-rich
sequences in the Ras GDP-GTP exchanger Son of Sevenless(18) .
Members of the ErbB family of RTKs and their ligands are implicated both in normal mammary gland development and the growth and progression of human breast cancer(19) . Furthermore, marked alterations in the expression or activity of several SH2 domain-containing proteins have been observed in human breast cancers or breast cancer-derived cell lines, suggesting that this represents an additional level at which RTK signaling may be modulated in this disease(20) . We therefore chose the human mammary epithelial cell line HMEC 184 as a model system on which to base a CORT screening program and hence identify novel, relatively tissue-specific, ErbB receptor targets.
Amino acid sequence alignments were performed using the computer programs Clustal W and SeqVu.
Figure 2: Determination of the GRB14 cDNA sequence. A, a schematic representation of GRB14 structure with a restriction map for the GRB14 cDNA and the cDNA clones used to derive the GRB14 sequence aligned below. The initial clone isolated by CORT screening was designated clone 1. Two other clones (1-1 and 1-2) were isolated from the 184 cell line library by screening using clone 1 as a probe. The GRB14 cDNA sequence was completed using two clones, L5 and L6, isolated from a human liver cDNA library. Abbreviations are as follows. A, ApaI, Av, AvrII, X, XhoI, E, EcoRI. The numbers refer to distance in bp. B, nucleotide and amino acid sequence of GRB14. The PH domain is underlined, and the SH2 domain is indicated by boldface type. The translation termination codon is shown by an asterisk in the amino acid sequence. Numbers refer to distances in bp.
A Flag epitope-tagged Grb14
eukaryotic expression vector was constructed as follows. The complete
open reading frame of GRB14 was first assembled in the vector
pRcCMV (InVitrogen Corp., San Diego, CA) using DNA restriction
fragments derived from cDNA clones L5 (in pBluescript SK+) and
1-2 (in pEXlox) (Fig. 2A). Briefly, L5 was
digested with SpeI (which cuts in the vector polylinker) and AvrII to delete a 93-bp fragment harboring an ApaI
site from the 5` end of this cDNA (Fig. 2A). The
digested vector was then religated, and a restriction fragment from
between the XbaI site in the polylinker and ApaI site
in the cDNA prepared and cloned into pRcCMV. The GRB14 open
reading frame was then completed by cloning an ApaI fragment
encoding the C-terminal region of Grb14 from clone 1-2 into this
vector. However, the resulting expression vector produced only low
yields of recombinant Grb14 in a coupled transcription/translation
system (Promega). Enhanced expression of Grb14 was achieved by deletion
of a GC-rich region 5` of the translation start codon. This was
achieved by synthesis of a cDNA encoding the GRB14 open
reading frame by polymerase chain reaction from the GRB14/pRcCMV template using oligonucleotide primers containing HindIII (5`) and BamHI (3`) sites for directional
cloning. This DNA fragment was inserted between the HindIII
and BglII sites of pRcCMV, a modified version
of pRcCMV designed for tagging of expressed proteins with the 8-amino
acid Flag epitope (DYKDDDDK) (24) at the C terminus. The
construction of pRcCMV
will be described in detail
elsewhere. The sequence of the GRB14 cDNA in this vector was
confirmed by DNA sequencing.
Growth factors were used at the following final concentrations: human recombinant EGF (Life Technologies, Inc., Glen Waverley, Victoria, Australia), 275 ng/ml; human recombinant PDGF BB (Life Technologies, Inc.), 50 ng/ml. HER14 and HER1-2 cells were stimulated with growth factors for 2 min at 37 °C, and HEK 293 cells were stimulated for 5 min at 37 °C.
The GST-Grb14 SH2
domain fusion protein was used to raise an anti-Grb14 polyclonal
antiserum in rabbits. The resulting antiserum (number 264) was
affinity-purified by an adaptation of the method of Smith and Fisher (29) using a fusion protein consisting of the Grb14 SH2 domain
fused to the T7 gene 10 product as an affinity reagent. This
methodology provided the additional advantage of removing anti-GST
antibodies in the same step. Briefly, the pEXlox plasmid containing GRB14 clone 1 (Fig. 2A) was excised and
transformed into E. coli BL21 DE3 pLysE. Lysates from
isopropyl--D-thiogalactopyranoside-induced bacteria were
separated by SDS-PAGE and transferred to nitrocellulose, and the
position of the induced fusion protein was identified by Ponceau S
staining. The filters were then incubated with crude antiserum diluted
1:1 in Tris-buffered saline, and following washing, the anti-Grb14
antibodies were eluted from the appropriate region of the filter by
incubation in 0.1 M glycine, pH 2.5, for 10 min. The eluate
was neutralized with 0.2
volume of 1 M Tris-HCl, pH
8.0, concentrated, and finally subjected to buffer exchange with
Tris-buffered saline (10 mM Tris-HCl, 150 mM NaCl, pH
7.4) using a Centricon 30 microconcentrator (Amicon, Beverly, MA). The
affinity-purified antibody was then quantitated, adjusted to 0.1%
bovine serum albumin, and stored at -70 °C.
Densitometric analysis of autoradiographs was performed using the IP Lab Gel analysis program (Signal Analytics Corp., Vienna, Virginia).
Direct binding of GST fusion proteins to growth factor receptors immobilized on nitrocellulose (Far Western blotting) was performed as described previously(30) , except that an anti-GST monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used for detection of bound fusion protein and antibody visualization was by ECL.
Stable transfection of HEK 293 cells was performed by the same protocol scaled up to 10-cm tissue culture dishes. Following transfection the cells were subcultured 1:10 and subjected to selection with Geneticin (1 mg/ml, Life Technologies, Inc.). Individual colonies were then isolated by trypsinization using cloning cylinders.
Figure 1:
Isolation of cDNA clones encoding EGFR
binding proteins by the CORT method. A EXlox cDNA expression
library prepared from the 184 normal human mammary epithelial cell line
was screened with the
P-labeled carboxyl terminus of the
EGFR. The figure shows tertiary screening results for cDNA
clones corresponding to Grb2, Ras-GAP/Grb13, and Grb14. The exposure
time for the autoradiographs was 4.5 h at -70 °C with one
intensifying screen.
Figure 4:
Northern blot analysis of Grb14 gene
expression. Northern blots of poly(A) RNA isolated
from a variety of human tissues were hybridized to a GRB14 cDNA probe labeled with
P by random primer extension.
The exposure time for the autoradiographs was 7 days at -70
°C with two intensifying screens.
Analysis of the cDNA sequence identified an open reading frame of 540 amino acids. The initiation codon is preceded by an in-frame termination codon and is surrounded by a consensus sequence for strong translational initiation(34) . The encoded protein is similar both in molecular architecture and amino acid sequence to Grb7 (2) and the recently identified Grb10(4) , consisting of an N-terminal region containing at least one proline-rich motif, a central region of approximately 320 amino acids, which exhibits significant homology to the C. elegans protein F10E9.6/mig 10 (4, 35) and which also encompasses a PH domain, and a C-terminal SH2 domain. An alignment of the amino acid sequences of Grb14, Grb7, Grb10 and F10E9.6 is shown in Fig. 3.
Figure 3: Sequence homology among Grb14, Grb7, Grb10, and F10E9.6. An alignment of the amino acid sequences of Grb14, mouse Grb7, mouse Grb10, and C. elegans F10E9.6 was obtained using the computer programs Clustal W and SeqVu. Identical residues are boxed. A highly conserved proline-rich motif is indicated by the dotted underline, the PH domain by the broken underline, and the SH2 domain by the thick underline. Only the central region of F10E9.6 exhibiting homology with the Grb7 family is shown. Amino acid residues for each protein are numbered (from the initiation methionine) on the right.
Grb14 is similar in size to Grb7, Grb10 possessing a more extended N terminus. The N-terminal regions of Grb14, Grb7, and Grb10 exhibit low sequence homology apart from a highly conserved amino acid motif P(S/A)IPNPFPEL. Also of note is the presence of two clusters of basic residues, which flank this motif. Overall, the N-terminal region of Grb14 displays a lower proline content than that of Grb7 and Grb10 (Grb14 amino acids 1-110, 11% proline; Grb10 amino acids 1-113, 15%; Grb7 amino acids 1-103, 23%).
In the conserved central region, Grb14 bears 48, 55, and 28% amino acid identity, respectively, with Grb7, Grb10, and F10E9.6 (Fig. 3). The core of this region is provided by a PH domain ( Fig. 2and Fig. 3)(7) , over which Grb14 exhibits 56, 61, and 35% amino acid identity, respectively, with Grb7, Grb10, and F10E9.6. However, as noted by Ooi et al.(4) , another region of marked homology spanning approximately 100 amino acids exists amino-terminal to the PH domain (Fig. 3).
The most highly conserved region among Grb7 family members is the SH2 domain (Fig. 3). The Grb14 SH2 domain displays 67 and 74% amino acid identity, respectively, with the corresponding domain in Grb7 and Grb10.
Since the GRB14 cDNA was originally isolated from a cDNA library prepared from normal human breast epithelial cells, we were interested in determining the expression profile of GRB14 mRNA in a panel of human breast cancer cell lines. Upon Northern blot analysis of total RNA isolated from 3 normal human breast epithelial cell strains and 19 human breast cancer cell lines, GRB14 gene expression could be detected in HMEC 184 and HMEC-219-4 cells, 6/7 ER+ human breast cancer cell lines, and 2/12 ER- cell lines (Table 1). Thus GRB14 gene expression appears largely restricted to normal breast epithelial and ER+ breast cancer cells. Differential expression of GRB14 was also observed among human prostate cancer cell lines. Although GRB14 mRNA expression was undetectable in the normal prostate (Fig. 4), low expression could be detected in the PC3 and LnCaP prostate cancer cell lines and high expression in the DU145 line (Table 1).
Figure 5: Expression of Grb14 protein in different human cell lines. A, detection of Grb14 by Western blot analysis. Lysates were prepared from the indicated cell lines and equivalent amounts of protein separated by SDS-PAGE (10% gels), transferred to nitrocellulose, and Western blotted with either preimmune serum or affinity-purified (A.P.) antiserum 264. Detection of bound antibody was by ECL. The mobility of molecular weight standards was as indicated. B, expression of Grb14 in human prostate cancer cell lines. Lysates were prepared from the indicated cell lines and equivalent amounts of protein subjected to Western blot analysis with affinity purified antiserum 264 as described previously.
Since DU145 cells overexpress GRB14 mRNA relative to the two other prostate carcinoma cell lines examined (Table 1), we investigated whether this was accompanied by an up-regulation of Grb14 protein expression. Upon Western blot analysis, Grb14 was clearly detectable in DU145, but not PC3 or LnCaP, cell lysates (Fig. 5B), indicating that Grb14 protein is overexpressed in this cell line.
When compared with either
the Grb7 (Fig. 6) or the Grb2 SH2 domains for
binding to the EGFR, the Grb14 SH2 domain exhibited a relatively weak
interaction, as might be predicted from the CORT screening results (Fig. 1). In accordance with other RTK-SH2 domain interactions,
the binding of the Grb14 SH2 domain was dependent on ligand stimulation
and tyrosine phosphorylation of the receptor. A difference in binding
selectivity between the Grb7 and Grb14 SH2 domains was more evident
when binding to the activated HER 1-2 receptor (an EGFR/ErbB2
chimera containing the intracellular domain of ErbB2) was investigated.
In this experiment the Grb7 SH2 domain bound avidly to the chimeric
receptor, as reported by Stein et al.(35) , while
binding of the Grb14 SH2 domain could not be detected (Fig. 6).
However, both the Grb7 and Grb14 SH2 domains exhibited strong binding
to activated PDGFRs (Fig. 6), which was more pronounced than
that observed with the Grb2 SH2 domain. (
)In Far Western
blotting experiments in which specific PDGFRs were immunoprecipitated,
separated by SDS-PAGE, transferred to nitrocellulose, and then
incubated with soluble GST-Grb14 SH2 domain, direct binding was
observed to both activated
- and
-PDGFRs.
Figure 6:
Interaction of the Grb14 SH2 domain with
different RTKs. Top panel, comparison of Grb14 SH2 domain
binding to the EGFR with that of Grb7. Lysates from either
control(-) or EGF-stimulated (+) HER14 cells were incubated
with GST, GST-Grb7 SH2, or GST-Grb14 SH2 coupled to glutathione-agarose
beads. Following washing, bound proteins were subjected to SDS-PAGE (8%
gel), transferred to nitrocellulose, and blotted with anti-EGFR
antibodies. Detection of bound antibodies was by ECL. Middle
panel, comparison of Grb14 SH2 domain binding to the HER1-2
receptor with that of the Grb7 SH2 domain. The experimental protocol
was as for the top panel except that HER1-2 cells were
used, and the detection of bound receptor was performed with an
anti-ErbB2 antibody. Lower panel, comparison of Grb14 SH2
domain binding to activated PDGFRs with that of the Grb7 SH2 domain.
The experimental protocol was as for the top panel except that
HER 14 cells were stimulated with PDGF BB, and the detection of bound
receptors was performed with anti-/
-PDGFR
antibodies.
The
interaction between Grb14 and RTKs in vivo was studied using
two systems: DU145 cells, which express high levels of endogenous Grb14
and 1.5 10
EGFRs/cell (36) and transient
transfection of HEK 293 cells.
Western blotting of EGFR
immunoprecipitates from EGF-stimulated DU145 cells with anti-Grb14
antiserum did not detect association between these two proteins.
Furthermore, upon transient co-expression of the EGFR and Flag
epitope-tagged Grb14 in HEK 293 cells, the EGFR could not be detected
in Grb14 immunoprecipitates from EGF-stimulated cells. Similarly,
following co-transfection of the
-PDGFR and GRB14 cDNAs into HEK 293 cells, Grb14 could not be detected in
anti-
-PDGFR immunoprecipitates from PDGF BB-stimulated cells, and
vice versa. Therefore, true in vivo binding partners for the
Grb14 SH2 domain remain to be identified.
Figure 7:
Characterization of Flag epitope-tagged
Grb14 expressed in HEK 293 cells. A, detection of
epitope-tagged Grb14 by Western blot analysis. Lysates were prepared
from control untransfected (HEK) and GRB14/pRcCMV transfected (HEK/Grb14) HEK 293 cells. Equivalent amounts of
protein were then separated by SDS-PAGE (10% gel), transferred to
nitrocellulose, and Western blotted with anti-Flag monoclonal antibody
M2. Detection of bound antibody was by ECL. B,
immunoprecipitation of epitope-tagged Grb14. Lysates were prepared from
control and transfected HEK 293 cells. Equivalent amounts of protein
were then immunoprecipitated (IP) with anti-Flag antibody M2
and subjected to SDS-PAGE (10% gel). Following transfer to
nitrocellulose the immunoprecipitates were Western blotted with
affinity-purified anti-Grb14 antiserum 264 or anti-Flag antibody M2. C, detection of Grb14 phosphorylation. Control and transfected
HEK 293 cells were serum-starved and then metabolically labeled with
P-orthophosphate. Following immunoprecipitation with
anti-Flag antibody M2 and SDS-PAGE, the immunoprecipitates were
transferred to a PVDF membrane and subjected to autoradiography. The
exposure time was 16 h at -70 °C with two intensifying
screens. D, detection of Grb14 serine phosphorylation.
Following detection of PVDF-immobilized phosphorylated Grb14 the band
was excised and subjected to phosphoamino acid analysis as described
under ``Materials and Methods.'' The mobilities of the
phosphoamino acid standards phosphotyrosine (Tyr),
phosphothreonine (Thr), and phosphoserine (Ser) were
as indicated. The exposure time was 14 days at -70 °C with
two intensifying screens. An equivalent of a more prolonged exposure
achieved using a PhosphorImager also only detected serine
phosphorylation.
Treatment of the cells with EGF did not
significantly increase this level of phosphorylation (Fig. 8A), although activation of native EGFRs could be
demonstrated by anti-phosphotyrosine blotting of the cell
lysates. However, stimulation with PDGF BB resulted in an
approximately 1.5-fold increase within 5 min of administration, and
transient transfection of a cDNA encoding
-PDGFRs into the cells
further amplified this response to approximately 2-fold (Fig. 8B). The small increase in phosphorylation that
occurred when this construct was present in the absence of PDGF BB was
presumably due to the constitutive activation of RTKs often observed
with this system(23) . Phosphoamino acid analysis demonstrated
that the PDGF-induced increases in Grb14 phosphorylation also occurred
on serine residues.
Figure 8:
Regulation of Grb14 serine
phosphorylation. A, regulation by EGF. HEK 293 cells
expressing Flag epitope-tagged Grb14 were serum-starved, metabolically
labeled with P-orthophosphate, and then either left
untreated(-) or stimulated with EGF (+) for 5 min at 37
°C. Following cell lysis, equivalent amounts of protein were
immunoprecipitated with anti-Flag antibody M2. Three-quarters of each
immunoprecipitate were separated by SDS-PAGE (8% gel), transferred to a
PVDF membrane and subjected to autoradiography (IP). The
remainder was Western blotted with affinity-purified anti-Grb14
antiserum 264 (IP, Blot). Following densitometric
analysis the amount of Grb14 phosphorylation was normalized for the
Grb14 content of each immunoprecipitate and expressed as a percentage
of the value for the untreated sample (lower panel). B, regulation by PDGF. HEK 293 cells expressing Flag
epitope-tagged Grb14 were transiently transfected with either vector
alone (lanes 1 and 2) or a
-PDGFR-encoding
expression vector (lanes 3 and 4). Following serum
starvation the cells were metabolically labeled with
P-orthophosphate and then either left untreated(-)
or stimulated with PDGF BB (+) for 5 min at 37 °C.
Determination of the relative amount of Grb14 phosphorylation was as
described previously.
This paper describes the expression cloning of a novel member
of a family of SH2 domain-containing signaling proteins which contains
two other proteins cloned by the CORT screening technique, Grb7 (2) and Grb10(4) . The cloning of GRB14 provides further evidence for the strength of the CORT screening
strategy in the identification of novel EGFR binding proteins. In
addition to the three members of the GRB7 gene family
characterized so far, utilization of this technique also led to the
cloning of GRB1/p85(5) and GRB2(3) .
Other SH2 domain-containing proteins isolated using this procedure
include phospholipase C-1, Fyn, Nck-like and Crk-like
proteins(2) , Syp(37) , and Ras-GAP (this manuscript).
Furthermore, a novel, non-SH2 Tyr(P) interaction (PI) domain in Shc was
also initially identified by this technique(38) .
Interestingly, CORT screening of the 184 cell line cDNA library also
led to the isolation of four cDNAs corresponding to a protein lacking
clearly recognizable SH2 or PI motifs.
The Grb7 family belong to the adapter subclass of SH2 domain-containing proteins and contain at least three ``interactive'' protein domains likely to participate in this function. First, the cloning of GRB14 has highlighted an N-terminal proline-rich motif P(S/A)IPNPFPEL, which is completely conserved in all three family members (Fig. 3). This is particularly striking when considered in the context of the poor sequence conservation elsewhere in the N termini of these proteins. The motif conforms to the consensus PXXP SH3 domain binding motif described by Yu et al.(6) , suggesting that recruitment of proteins containing a particular subclass of SH3 domain is fundamental to Grb7 family signaling. Interestingly, Grb7 and Grb10 also contain other proline-rich regions harboring PXXP motifs that may provide additional SH3 domain binding sites; however, these are not conserved between the two proteins.
The second region probably involved in intermolecular interaction is the central region of approximately 320 amino acids bearing homology to the C. elegans protein F10E9.6. A key feature of this region is a PH domain, which may mediate protein-protein or protein-phospholipid interactions (7, 15, 39) and hence regulate signaling events and/or subcellular localization. However, since homology with F10E9.6 extends outside the PH domain (in particular to the 100-amino acid region amino-terminal to this domain) it seems likely that other sections of the central region also participate in a conserved signaling function. The only clue to the role of this region comes from the recent identification of F10E9.6 as the product of the mig10 gene in C. elegans(4, 35) , which is required for longitudinal neuronal migration in embryos(40) . However, Grb7 family members and F10E9.6/mig 10 do not exhibit overall structural similarity, the latter containing an unrelated N-terminal region and a proline-rich C terminus flanking the central domain. It therefore remains possible that this central domain represents a protein module found in functionally distinct proteins and that the Grb7 family and F10E9.6/mig 10 perform unrelated signaling roles.
The third interactive domain identified in Grb14 is the SH2 domain.
When expressed as a GST fusion protein and utilized for in vitro binding experiments, this domain exhibited a high affinity for a
subclass of tyrosine-phosphorylated growth factor receptors, binding
strongly to activated PDGF receptors in HER14 cells but only weakly to
activated EGF receptors (Fig. 6). Interaction with an EGFR-ErbB2
chimera was undetectable. This represents a marked difference in
binding specificity to the Grb7 SH2 domain, which binds strongly in
vitro to the EGF and ErbB receptors(35, 38) . The
Grb10 SH2 domain, which is more closely related to the Grb14 SH2 domain
than that of Grb7, also displays a relatively weak interaction with the
EGFR(4) . This gene family therefore represents an interesting
model system in which to study determinants of SH2 domain binding
selectivity. Indeed, we have recently identified two amino acid
residues in Grb14 that, when changed to their Grb7 counterparts, confer
high affinity in vitro binding to ErbB2. ()However,
we have not detected association between either the EGFR or
-PDGFR
and Grb14 in vivo, even upon transient co-expression in HEK
293 cells. Binding of the Grb14 SH2 domain to tyrosine-phosphorylated
receptors/proteins may therefore be restricted by the subcellular
localization, conformation, and/or phosphorylation of the full-length
protein as well as the inherent binding selectivity of this domain.
Recently, two potential in vivo partners for the Grb10 SH2
domain, the insulin and Ret RTKs, were identified by two-hybrid
screens(41, 42) , and the interaction of these
proteins with Grb14 is currently under investigation.
Although in vivo association between the -PDGFR and Grb14 could
not be demonstrated, a role for Grb14 in signaling events initiated by
this receptor class was demonstrated by the increase in Grb14 serine
phosphorylation observed upon PDGF BB activation of native PDGF
receptors or transfected
-PDGFRs (Fig. 8B).
Interestingly, Grb10 also exhibits a basal level of serine
phosphorylation that increases upon PDGF treatment without detectable
PDGFR recruitment(4) . However, the phosphorylation of Grb10,
but not of Grb14, also increases in response to EGF
stimulation(4) . Whether this represents a difference in
signaling specificity between these two family members or reflects the
different cell types utilized in these experiments is unknown at
present. The growth factor-induced serine phosphorylation of Grb10 was
not mimicked by phorbol ester treatment (4) , demonstrating
that signaling via conventional or novel protein kinase C isoforms was
not involved(43) . Since PDGF is more potent at increasing
Grb14 phosphorylation than treatment with
12-O-tetradecanoylphorbol-13-acetate,
this
strongly suggests that signaling via phorbol ester-activated protein
kinase C isoforms is also not the major mechanism for PDGF-induced
Grb14 phosphorylation. Determination of the sites of phosphorylation of
Grb14 and their degree of conservation among the Grb7 family will
provide further insight into the role of phosphorylation in regulation
of the function of these proteins. Also, further characterization of
the serine kinase involved and its interaction with Grb14 will also
help determine whether this activity represents an integral component
of the Grb14 signaling complex.
The GRB7 gene family exhibit relatively tissue-specific patterns of expression. GRB14 probably represents the most widely expressed family member and GRB7 the least, expression of the latter gene being restricted to kidney, liver, and gonads(2) . These proteins therefore differ from other ``adapter'' SH2 domain-containing proteins such as Grb2 (3) and Nck(44) , which are ubiquitous in their expression. This suggests that the Grb7 family performs a relatively specialized signaling role, with the individual members functioning in a tissue-restricted manner to link specific receptors to effector molecules. This hypothesis is supported by the differences in SH2 domain specificity exhibited by the different family members. However, Grb7 family proteins also exhibit a provocative expression profile among different types of human cancer cells. For example, due to the close proximity of their respective genes and hence coordinate gene amplification, Grb7 is overexpressed along with its binding partner ErbB2 in a subset of human breast cancers(35) . Furthermore, our results demonstrate a correlation between GRB14 expression and estrogen receptor positivity in human breast cancer cells and, in a preliminary investigation of human prostate cancer cell lines, marked overexpression in DU145 cells versus the normal prostate and the other prostate cancer lines examined ( Table 1and Fig. 5). These and other data demonstrating aberrant expression of signaling proteins in human cancer cells (20) identify novel mechanisms for modulation of RTK signaling during tumorigenesis and highlight a potential role for such proteins as tumor markers and/or prognostic indicators.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L76687[GenBank].