From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Integrin Integrins are a family of transmembrane cell adhesion receptors
composed of Four different We previously investigated the function of the In this study, we used Cell Culture--
Mouse fibroblast C3H 10T1/2 cells were grown
in Dulbecco's modified Eagle's medium low glucose (Life Technologies)
supplemented with 10% fetal bovine serum (Life Technologies) and
glutamine/penicillin/streptomycin (Life Technologies). The human
prostate epithelial cell line DU145 was grown in Dulbecco's modified
Eagle's medium high glucose supplemented with
glutamine/penicillin/streptomycin, nonessential amino acids (Sigma),
and 10% fetal bovine serum. The human endothelial cell line EV304 was
grown in endothelial growth media (Clonetics) supplemented with 10%
fetal bovine serum. C3H 10T1/2 cells were transiently transfected using
LipofectAMINE (Life Technologies) as per the manufacturer's
instructions. DU145 and EV304 cells were transiently transfected using
Effectene (Qiagen) as per the manufacturer's instructions. DNAs for
transfection were prepared using Plasmid Maxi Kits (Qiagen).
DNA Constructs--
IL2 receptor integrin chimeras were
generated by fusing various integrin Flow Cytometry--
Cell growth in transiently transfected cells
was measured by labeling cells with
bromodeoxyuridine/fluorodeoxyuridine (BrdUrd; Amersham) for 16 h
starting at 32 h after transfection. Labeled cells were then
detached with trypsin/EDTA (Life Technologies), washed once with growth
media and twice with 1% bovine serum albumin/PBS (nuclease- and
protease-free; Calbiochem), and then fixed in cold 70% ethanol on ice
for 30 min. Cellular DNA was denatured by treating fixed cells with 2 N HCL/0.5% Triton for 30 min and stopped by incubating
cells in 0.1 M Na2B4O7,
pH 8.5. Cells were then washed once with 0.5% Tween 20/1% bovine
serum albumin/PBS (TBP). IL2 receptor chimera expression was determined
by incubating cells with a phycoerythrin-conjugated anti-IL2 receptor
antibody (PharMingen), and BrdUrd incorporation was determined by using
a fluorescein isothiocyanate-conjugated anti-BrdUrd antibody (Becton
Dickinson). Antibodies were diluted in TBP buffer, and labeling
reactions were incubated at 37 °C for 30 min. Labeled cells were
analyzed using a Becton Dickinson FACScan and CellQuest software.
Approximately 100,000 cells were analyzed per construct in each
experiment. BrdUrd incorporation was determined for different levels of
chimera expression, with expression level gates set such that at least 1,000 cells were included in each gate. BrdUrd incorporation levels were determined, normalized against the IL2R/TL control, and expressed as the percentage of inhibition per mean expression level.
Immunofluorescence--
Transiently transfected cells were
detached with trypsin/EDTA and replated in growth medium on glass
coverslips 24 h after transfection. Cell growth was measured by
labeling cells with BrdUrd for 5 h starting at 48 h after
transfection. Labeled cells were fixed in 2% formaldehyde (EM
Sciences)/PBS for 15 min at room temperature, extracted with 0.2%
Triton/PBS for 10 min, treated with 0.1 unit/µl DNase I for 30 min at
37 °C, and blocked in 10% normal goat serum/PBS (Life
Technologies). Primary and secondary antibodies were diluted in 10%
normal goat serum/PBS, and labeling reactions were incubated for 30 min
at 37 °C. In order to measure BrdUrd incorporation in cells
expressing IL2 receptor chimeras, cells were probed with an anti-BrdUrd
primary antibody (Calbiochem) followed by a fluorescein
isothiocyanate-conjugated goat anti-mouse IgG secondary antibody
(Jackson ImmunoResearch). Cells were then blocked with mouse IgG
(Sigma) before labeling with a phycoerythrin-conjugated anti-IL2
receptor antibody (PharMingen). In order to measure BrdUrd incorporation in cells expressing GFP chimeras, cells were probed with
an anti-BrdUrd primary antibody followed by a lissamine rhodamine sulfonyl chloride-conjugated goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch). Coverslips were mounted in Immunofluore mounting media (ICN) and examined using a Leitz diaplan fluorescence microscope. Fluorescent images were obtained using a Bio-Rad 1024 MRC
Scanning Confocal Microscope.
Localization of Integrin Cytoplasmic Domain-IL2 Receptor
Chimeras--
To map the growth regulatory sequences in
Effects of IL2R Chimeras on DNA Synthesis--
We next tested the
effects of the different chimeras on DNA synthesis. Transiently
transfected cells were replated on glass coverslips after 24 h and
incubated with the thymidine analogue BrdUrd for 5 h starting at
48 h after transfection. Cells were then fixed and double-stained
for IL2R expression and BrdUrd incorporation. We have found that high
levels of chimera expression induced cell retraction and rounding and
inhibited cell adhesion or
spreading,3 consistent with
previous reports that these chimeras can function as dominant negative
inhibitors for endogenous integrin function (20, 23, 26). Because the
10T1/2 fibroblasts are anchorage-dependent, effects on cell
adhesion would result in nonspecific growth arrest. Therefore, to rule
out this type of effect, only morphologically normal cells were scored
for nuclear labeling with anti-BrdUrd. As shown in Fig.
2, the IL2R/ Dose-Response Curves--
To obtain a more quantitative estimate
of the dependence of growth inhibition on expression levels,
dose-response curves for the
The results in Fig. 3 using fluorescence-activated cell-sorting
analysis show significant inhibition of DNA synthesis at high levels of
Mapping Inhibitory
In order to further define growth regulatory sequences, we generated a
series of IL2R/ Growth Inhibition in Human Cell Lines--
Previous reports
suggested that We used chimeras of the 1C is an
alternatively spliced cytoplasmic variant of the
1 subunit that
potently inhibits cell cycle progression. In this study, we analyzed
the requirements for growth suppression by
1C. A chimera
containing the extracellular/transmembrane domain of the Tac subunit of
the human interleukin 2 receptor (gp55) fused to the cytoplasmic domain
of
1C (residues 732-805) strongly inhibited growth in
mouse 10T1/2 cells even at low expression levels, whereas chimeras
containing the
1A,
1B,
1D,
3, and
5 cytoplasmic domains had weak and
variable effects. The
1C cytoplasmic domain is composed
of a membrane proximal region (732-757) common to all
1
variants and a COOH-terminal 48-amino acid domain (758-805) unique to
1C. The
1C-specific domain (758-805) was sufficient to block cell growth even when expressed as a soluble cytoplasmic green fluorescent protein fusion protein. These results indicate that growth inhibition by
1C does not require
the intact receptor and can function in the absence of membrane
targeting. Analysis of deletions within the
1C-specific
domain showed that the 18-amino acid sequence 775-792 is both
necessary and sufficient for maximal growth inhibition, although the 13 COOH-terminal residues (793-805) also had weak activity. Finally,
1C is known to be induced in endothelial cells in
response to tumor necrosis factor and is down-regulated in prostate
epithelial cells after transformation. The green fluorescent
protein/
1C (758-805) chimera blocked growth in the
human endothelial cell line EV304 and in the transformed prostate
epithelial cell line DU145, consistent with a role for
1C as a growth inhibitor in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
and
subunits; 12 different
subunits and 9 different
subunits have been identified to date (reviewed in Refs.
1 and 2). The
1 subunit pairs with
1,
2,
3,
4,
5,
6,
7,
8,
9,
and
v to generate 10 different receptors that show
distinct binding specificities for extracellular matrix proteins and
other cell surface receptors. In addition to their role as adhesion
receptors, integrins regulate cellular functions including cytoskeletal
organization, cell growth, gene expression, survival, and migration.
These effects are dependent on signaling events involving changes in
protein phosphorylation, protein interactions, intracellular ion
concentrations, and lipid metabolism. Integrin cytoplasmic domains lack
intrinsic enzymatic activity but have been shown to activate a variety
of signaling pathways including protein kinases (focal adhesion kinase,
integrin-linked kinase, protein kinase C, and mitogen-activated protein
kinase), lipid kinases, phospholipases, and small GTP-binding proteins
(Ras, Rac, and Cdc 42) and to bind to cytoskeletal proteins including
-actinin, talin, filamin, and paxillin.
1 cytoplasmic domain isoforms have been
identified (
1A,
1B,
1C,
and
1D), and at least one is expressed in nearly every
cell type (3-6). The
1A isoform is the primary isoform
in most cells except for skeletal muscle, where
1D
predominates (4, 5). These isoforms are generated by alternative
splicing that occurs between the sixth and seventh exons (7). As a
result, each of the
1 cytoplasmic domains contains a
common 26-aa1 membrane
proximal sequence and a unique COOH-terminal sequence. The potential
significance of these splice variants is highlighted by the fact that
many integrin-regulated signals are mediated by the
subunit
cytoplasmic domain. For example, the
1A cytoplasmic domain has been shown to interact with the cytoskeletal proteins
-actinin, filamin, and talin, and sequences within the unique
1A domain are required for focal adhesion localization
(8-15). Taken together, these observations suggest that alternative
splicing within the
1 gene may have profound effects on
receptor function.
1C
isoform in mouse 10T1/2 fibroblasts. We found that expression of
1C induced a potent inhibition of cell cycle
progression, leading to arrest in late G1 (16).
1C failed to localize to focal adhesions, suggesting
that it has altered interactions with cytoskeletal proteins (16).
1C also blocked the growth of Chinese hamster ovary
cells (17). The functions of
1C in vivo are
unknown, but
1C expression has been shown to correlate
with growth arrest. It is up-regulated in human umbilical endothelial
cells in response to treatment with tumor necrosis factor, which blocks
the growth of these cells (17), and was found in quiescent prostate
epithelial cells but was down-regulated in prostate carcinoma,
suggesting that it could function as a tumor suppressor (18).
1C chimeras to define the
sequences necessary and sufficient for regulating growth. We identified a critical 18-aa region and showed that
1C sequences can
function apart from the rest of the integrin, even in the absence of
membrane targeting. We also found that the
1C-specific
domain blocks DNA synthesis in endothelial and prostate carcinoma
cells, suggesting that
1C protein may inhibit growth
in vivo.
MATERIALS AND METHODS
subunit cytoplasmic domains to
the extracellular/transmembrane domain of the IL2 receptor
subunit
(gp55) using the unique membrane proximal HindIII site.
Chimeras were expressed under the control of the cytomegalovirus
promoter in pCDNA3 expression vector (Invitrogen). Cytoplasmic
domains and cytoplasmic domain fragments of the integrin
1A,
1C,
1D, and
5 subunits were generated by polymerase chain reaction
amplification using the pBJ
1A, pBJ
1C,
pC
1D, and pBJ
5 cDNAs, respectively.
The full-length
1B cytoplasmic domain was generated by
polymerase chain reaction using overlapping primers and
pBJ
1A cDNA. All fragments were subcloned as
HindIII/XbaI fragments containing an in-frame
NH2-terminal HindIII site and a COOH-terminal
stop codon followed by a XbaI site. Construction of the IL2
receptor chimeras lacking a cytoplasmic domain (IL2R/TL) or containing
the integrin
3 cytoplasmic domain
(IL2R/
3) or the full-length
1D
cytoplasmic domain
(IL2R/
1D)2 has
been described previously (19, 20). The GFP/
1C chimera was generated by polymerase chain reaction amplification of the
1C-specific domain 758-805 containing an in-frame
NH2-terminal EcoRI site and a COOH-terminal stop
codon followed by a SalI site and subcloned into the
EcoRI/SalI sites of the GFP expression vector
pEGFP-C1 (CLONTECH). Polymerase chain reaction
primers were purchased from Life Technologies. All constructs were
verified by DNA sequencing. The protein numbering for
1C
is based on the mature protein such that the first residue of the
1C-specific domain is 758; other investigators have used
a numbering system based on the immature protein such that the
1C-specific domain begins with 778 (17).
RESULTS
1C, we generated a chimera containing the
extracellular/transmembrane domain of the 55-kDa Tac subunit of the IL2
receptor fused to the cytoplasmic domain of
1C
(IL2R/
1C). Similar chimeras have been used to
investigate the signaling of other integrin
subunit cytoplasmic
domains, including
1A,
1D, and
3 (19-24). These chimeras have been shown to mimic
properties of wild type receptors, including focal adhesion localization and the activation of focal adhesion kinase
phosphorylation, but in a ligand-independent manner (21). Mouse 10T1/2
fibroblasts were transiently transfected with IL2 receptor chimeras
containing the
1A,
1B,
1C,
and
1D cytoplasmic domains and stained for surface
expression. A tail-less construct (TL) that did not contain any
integrin sequences was used as a control. As shown in Fig. 1, IL2R/
1C
(
1C) exhibited a diffuse staining pattern
similar to the staining pattern of the tail-less IL2R chimera
(TL). This result is consistent with the diffuse staining
pattern observed for the full-length receptor (16). The
IL2R/
1B chimera (
1B) also
displayed a diffuse staining pattern similar to the wild type
1B receptor (25). In contrast to the
1C
and
1B chimeras, the
1A and
1D chimeras were localized to focal adhesions, in agreement with previous reports (19, 20, 24).
View larger version (36K):
[in a new window]
Fig. 1.
Localization of the IL2 receptor
chimeras. Mouse 10T1/2 cells were transiently transfected with
expression vectors coding for the IL2 receptor extracellular and
transmembrane sequence lacking a cytoplasmic domain (TL) or
fused to the cytoplasmic domains of the integrin 1
splice variants
1A,
1B,
1C, or
1D. Cells were fixed and stained
for surface expression using a phycoerythrin-conjugated anti-IL2
receptor antibody. Bar, 10 µm.
1C chimera
blocked BrdUrd incorporation in >99% of the expressing cells. In
contrast, the IL2R/
1A,
1B, and
1D chimeras showed varying but relatively modest effects on BrdUrd labeling. The
1A and
1B
chimeras caused decreases in DNA synthesis, whereas the
1D chimera induced a modest increase compared with the
IL2R/TL control (Fig. 2). We also examined chimeras containing the
integrin
3 and
5 cytoplasmic domains,
both of which localized to focal adhesions.3 As shown in
Fig. 2, the
3 chimera induced an increase in cells labeled with BrdUrd, whereas the
5 chimera partially
inhibited labeling. These results indicate that only the
1C cytoplasmic domain functions as a potent inhibitor,
although the other
subunit cytoplasmic domains have detectable
effects on growth. These data also indicate that the
1C
cytoplasmic domain is sufficient to induce cell cycle arrest when
separate from the rest of the integrin.
View larger version (17K):
[in a new window]
Fig. 2.
Effect of IL2 receptor chimeras on cell
growth. Mouse 10T1/2 fibroblasts were transiently transfected with
vectors for IL2 receptor chimeras with no cytoplasmic domain
(TL) or containing the cytoplasmic domains for the integrins
1A,
1B,
1C,
1D,
3, and
5. Transfected
cells were replated on glass coverslips 24 h after transfection
and labeled with BrdUrd for 5 h at 48 h after transfection.
Cells were fixed and double-stained for IL2 receptor expression and
BrdUrd incorporation. Cells positively stained with anti-IL2R were
scored for BrdUrd labeling. 100 cells were scored per construct per
experiment. Values are the means ± S.D. of three
experiments.
1A and
1C
chimeras were generated. Starting at 30 h after transfection,
cells were labeled with BrdUrd for 16 h and then detached, fixed,
and stained for both BrdUrd incorporation and IL2R chimera expression.
Cells were then assayed by flow cytometry. Using this protocol, the
maximum growth inhibition was 45-50%; even cells that were detached
and put into suspension at the beginning of the labeling period were
inhibited by only 45%. Presumably, this result reflects the fact that
a significant fraction of the cells have passed the restriction point
and are already committed to or have entered S phase. The correlation
between expression level for IL2R and BrdUrd staining is shown in Fig.
3. We found that both of the integrin
chimeras induced growth inhibition, although at markedly different
expression levels. The
1A chimera blocked growth at high
expression levels, consistent with the idea that this chimera may
function as dominant negative inhibitor for endogenous integrin
function. In contrast, the
1C chimera was a distinctly
more potent, maximally inhibiting DNA synthesis at a level of
expression just above background.
View larger version (18K):
[in a new window]
Fig. 3.
Dose-response analysis of
IL2R/ 1A and IL2R/
1C. Mouse 10T1/2
fibroblasts were transiently transfected with the IL2 receptor chimeras
IL2R/TL, IL2R/
1A, or IL2R/
1C. Cells were
labeled with BrdUrd for 16 h at 32-48 h after transfection,
detached, fixed, and double-stained for IL2R chimera expression and
BrdUrd incorporation. Approximately 106 labeled cells per
construct were analyzed by flow cytometry. BrdUrd incorporation was
determined for different levels of chimera expression and expressed as
the percentage of inhibition per mean expression level relative to the
IL2R/TL control. The results from one experiment are shown; similar
results were seen in five independent experiments.
1A expression, in disagreement with Fig. 2, in which DNA
synthesis was only slightly decreased in
1A-expressing
cells scored after plating on coverslips. One possible source of this discrepancy is that high expressors might be lost after replating and
their influence might be further decreased because rounded cells were
not scored. To investigate this question, cells transfected with
1A or
1C were left in the original dish
or replated into a fresh tissue culture dish and then detached and
analyzed for expression of the transfected integrins. Replating caused
a substantial loss of the
1A high expressors from the
population, whereas no decrease in
1C-expressing cells
was observed.3 This result explains the difference between
the two methods and supports the conclusion that growth inhibition by
1A is due to a dominant negative effect on cell
adhesion, whereas inhibition by
1C is caused by a
distinct mechanism.
1C Sequences--
The
1C cytoplasmic domain (residues 732-805) is composed of
a common region (732-757) shared with the other
1
splice variants and a unique region (758-805) specific to
1C. Hungerford et al. (27) showed that
microinjection of a peptide derived from the common
1
region can block endogenous integrin function and induce programmed
cell death in chicken fibroblasts in serum-free media. As shown in Fig.
4, deletion of the common region had no
effect on growth inhibition by
1C, indicating that the
1C-specific domain was sufficient. We next asked whether
the
1C-specific domain could block growth when expressed
as a soluble protein in the absence of membrane targeting. A chimera
was generated containing the
1C-specific domain
(758-805) fused to the GFP. This chimera blocked growth in 93% of the
expressing cells (Fig. 4B). Thus, growth inhibition by
1C occurs even when expressed as a soluble cytoplasmic
protein.
View larger version (17K):
[in a new window]
Fig. 4.
Effect of the 1C-specific
domain (758-805) on cell growth. Mouse 10T1/2 fibroblasts were
transiently transfected with various IL2 receptor chimeras
(A) or GFP chimeras (B) and assayed for cell
cycle progression. Transfected cells were replated on glass coverslips
24 h after transfection and labeled with BrdUrd for 5 h at
48 h after transfection. Cells were then fixed and analyzed for
chimera expression and BrdUrd incorporation. Values are the means ± S.D. of three independent experiments.
1C chimeras containing deletions within the
1C-specific domain (758-805). Deletion of the
membrane-proximal 17 aa (Fig. 5,
construct 2) had no effect on growth inhibition, whereas the deletion
of an additional 8 aa (construct 3) led to a substantial increase in
DNA synthesis. Expression of the COOH-terminal 13 aa alone (construct
4) had weak but reproducible growth-inhibitory activity. When we
screened deletions from the COOH terminus of the
1C-specific domain, we found that deletion of the
COOH-terminal 13 aa failed to increase DNA synthesis (constructs 2-4).
This result was surprising, because we had previously reported that the
deletion of this COOH-terminal 13 aa region (793-805) abolished growth
inhibition by the intact receptor (16). Deletion of the next 10 residues (Fig. 5B, construct 5) almost completely eliminated growth inhibition. Taken together, these results identify an 18-aa region, 775-792, that is the major site of the growth-inhibitory activity. The COOH-terminal 13-aa region also has weak activity; curiously, it is required for growth inhibition by the intact receptor
but not by the shorter
1C-specific domain expressed as a
chimera with the IL2R. This sequence may therefore be important for the
proper conformation of the intact receptor but not a shorter peptide.
View larger version (21K):
[in a new window]
Fig. 5.
Effects of deletions within the
1C-specific sequence. Mouse 10T1/2 fibroblasts were
transiently transfected with IL2 receptor chimeras containing various
NH2-terminal (A) or COOH-terminal (B)
deletions within the
1C-specific domain (758-805).
Transfected cells were replated on glass coverslips 24 h after
transfection and labeled with BrdUrd for 5 h at 48 h after
transfection. Cells were than fixed and double-stained for IL2R chimera
expression and BrdUrd incorporation. IL2 chimera expressors were scored
for the number of cells labeled with BrdUrd, and the percentage of
BrdUrd incorporation was normalized against the percentage of
incorporation for IL2R/TL. 100 cells were scored per construct per
experiment. Values are the means ± S.D. of three independent
experiments.
1C may function as a cell growth
regulator: its expression is induced in human umbilical endothelial
cells in response to tumor necrosis factor, which causes growth arrest,
and
1C is down-regulated during the transition from
normal, quiescent prostate epithelium to prostate carcinoma (17, 18).
To test the ability of
1C to block growth in these cell
types, GFP/
1C was transiently expressed in the human
endothelial cell line EV304 and in the transformed prostate epithelial
cell line DU145. Expressing cells were labeled with BrdUrd to assay cell cycle progression. As shown in Fig.
6, GFP/
1C induced growth arrest in both the EV304 cells and the DU145 cells, blocking BrdUrd labeling by 46.7% and 72.8%, respectively, compared with GFP
controls. These results indicate that
1C can block cell
growth in human cell lines relevant to its in vivo
expression.
View larger version (16K):
[in a new window]
Fig. 6.
Effect of GFP/ 1C on the growth
of endothelial cells and prostate epithelial cells. Human
endothelial cells (EV304) (A) or transformed human prostate
epithelial cells (DU145) (B) were transiently transfected
with vector, GFP, or GFP/
1C constructs. Transfected
cells were replated on glass coverslips 24 h after transfection
and labeled with BrdUrd for 5 h at 48 h after transfection
and then fixed and stained for BrdUrd incorporation. Total transfected
cells (vector) or GFP-expressing cells (GFP;
GFP/
1C) were scored for the percentage of cells labeled
with BrdUrd. 100 cells were analyzed per construct per experiment.
Values are the means ± S.D. from three independent
experiments.
DISCUSSION
1C cytoplasmic domain with
both the IL2 receptor and GFP to map growth regulatory sequences in
integrin
1C. The data revealed that growth inhibition by
1C occurred at low expression levels and was mediated
entirely by the
1C-specific domain. This domain
functioned in the absence of the
subunit extracellular,
transmembrane, and common cytoplasmic sequences, did not require the
subunit, and was effective in the absence of membrane targeting.
Additional deletion mapping showed that the primary activity resided
within an 18-aa region (775-792). The
1C cytoplasmic
domain also blocked growth when expressed in human endothelial cells
and transformed prostate epithelial cells, suggesting that the
endogenous protein may function in a similar capacity under
physiological conditions. These conclusions are summarized in Fig.
7.
View larger version (27K):
[in a new window]
Fig. 7.
Model of the 1C signaling
domain. Deletion analysis indicates that the 18-aa region
(775-792; box) within the
1C-specific domain
is necessary and sufficient for inducing growth arrest. The
COOH-terminal 13-aa region (793-805; underline) also
induces partial growth arrest and is required for growth inhibition by
the intact receptor (16). These results suggest that this region may
contain a secondary effector region and may also be required for
inducing the proper conformation within the full-length receptor. The
region identified by Fornaro et al. (775-782;
underline) (17) is also indicated. The protein sequences of
four Alu-containing proteins that are homologous to
1C
are shown below: an alternatively spliced variant of the
neurofibromatosis 2 tumor suppressor gene (NF2), an
alternatively spliced variant of the c-myb transcription factor
(c-myb), the transformation-related protein
(TRP), and the neuronal thread protein AD7c-NTP
(NTP). The predicted protein sequence of an Alu-Sx element
in the antisense orientation is also shown.
Other integrins had some effects on cell cycle progression. The
1A chimera decreased DNA synthesis at high levels,
consistent with reports that it functions as a dominant negative
inhibitor for endogenous integrin function in cell adhesion (20, 23, 26). Expression of
1B also decreased DNA synthesis,
consistent with published data indicating that this isoform can
function as a dominant negative inhibitor (28). We observed some
stimulation of DNA synthesis by
1D, whereas Belkin and
Retta (24) reported that the
1D chimera blocks cell
growth when expressed in C2C12 myoblasts, suggesting that effects of
1D may be cell type-specific.
Two previous studies reported the effect of COOH-terminal deletions
within the full-length 1C receptor on DNA synthesis (16, 17). We found that the COOH-terminal 13-aa region was required for
growth inhibition by the full-length receptor in 10T1/2 cells, whereas,
using a different protocol, Fornaro et al. (17) reported that a receptor lacking this region blocked growth in Chinese hamster
ovary cells. The results reported here may help resolve this
discrepancy by showing that the last 13 aa are not required for growth
inhibition by a shorter IL2 receptor chimera that contains only the
1C-specific sequences. Thus, this region may be required for maintaining proper conformation in some contexts, but it does not
appear to be essential for signaling.
Our findings and those of Fornaro et al. (17) both indicate
that the 775-782 region is critical; however, we found that this
region was not sufficient for growth inhibition but also required
residues 783-792. By contrast, Fornaro et al. (17) observed
growth inhibition in a construct lacking residues COOH-terminal to 782. This difference could be due to the difference in cell type or the
distinct protocol used in their studies, in which expressing cells were
analyzed after being captured by panning on anti-1 antibodies.
Alternatively, those studies used full-length
1C
constructs that contained the common, membrane-proximal region. Dominant negative effects leading to growth inhibition could therefore complicate the interpretation of those results.
The 1C-specific exon is formed by an Alu sequence in the
antisense orientation located between the sixth and seventh exon of the
human
1 gene (3, 29). Alu sequences are members of the short
interspersed nucleotide elements family of repetitive elements, and
approximately 700,000 Alu elements are thought to be present per
haploid human genome; thus, an average of more than 1 Alu element is
predicted to be present in every gene (30). Many genes express
Alu-containing sequences, and a few Alu-containing proteins have been
reported previously (31). The function or significance of these
elements is not known. We have mapped the
1C growth
inhibitory domain to a region of
1C that overlaps a
sequence that is conserved in other Alu-containing proteins including
transformation-related protein and neuronal thread protein and
alternatively spliced isoforms of neurofibromatosis 2 and c-myb (Fig.
7). The function of these proteins and their roles in growth control
are not known. In addition to this conserved region, the
1C growth inhibitory domain also contains a TSR sequence that is not found in the other Alu-containing proteins (Fig. 7).
The design of cell growth inhibitors is one of the major strategies in
the fight against cancer and other human diseases. We have identified a
short peptide sequence that is sufficient to induce growth arrest in
endothelial cells (EV304), in transformed prostate epithelial cells
(DU145), and in the K562 transformed hematopoietic cell line (data not
shown) as well as in rodent fibroblast cell lines. These results
suggest that the identification of 1C-binding proteins
and the characterization of
1C-mediated downstream
signaling pathways could aid in the development of novel angiogenesis
or tumor growth inhibitors.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Mark Ginsberg for generously
providing us with the IL2R/1D expression plasmid and
Bette Cessna for administrative assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants P01 HL48728 (to M. A. S.), R01 GM47214 (to M. A. S.), and GM47157 (to Y. T.) and by National Institutes of Health Training Grant T32 HL07695.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.
A research fellow of the American Heart Association, California Affiliate.
§ To whom correspondence should be addressed: Dept. of Vascular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-784-7137; Fax: 619-784-7360; E-mail: schwartz{at}scripps.edu.
2 D. Calderwood, C. Fenczik, and M. H. Ginsberg, manuscript in preparation.
3 J. Meredith and M. A. Schwartz, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: aa, amino acid(s); IL2, interleukin 2; IL2R, IL2 receptor; GFP, green fluorescent protein; BrdUrd, bromodeoxyuridine; PBS, phosphate-buffered saline.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|