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INTRODUCTION |
Integrins are cell surface receptors that are involved in
cell-matrix adhesion and signaling (recently reviewed in Ref. 1). The
6 integrin is a laminin receptor and contains 1050 amino acids present as a heavy (110 kDa) and a light (30 kDa) chain that are
linked by a disulfide bond (2). The heavy chain of
6
integrin contains an 875-amino acid extracellular region and interacts
with the
subunit to form the heterodimer (3). All the described
integrin
subunits contain seven weak sequence repeats in the
N-terminal region that are thought to be important in ligand binding
and have been predicted to fold cooperatively into a single
-propeller domain with seven
-sheets (4, 5). The minimum
essential elements of the extracellular domains for subunit pairing and
ligand binding are of considerable interest in understanding integrin
regulation (6). Therefore, we have extended our previous studies, which
indicated that a smaller variant of the
6 integrin
exists (7, 8).
Two alternatively spliced forms of
6 exist, containing
identical heavy chains and different light chains known as
6A and
6B (3, 9). The light chain of
6 integrin contains 170 amino acids composing an
extracellular region, the transmembrane region, and the cytoplasmic
domain (3). The
6A or
6B integrin subunit
can pair with either the
1 or the
4
subunit (10) and is found on a variety of normal cell types. It is
found on platelets (11), epithelia (12-15), endothelia (10, 16, 17),
proximal and distal tubules of the kidney (18, 19), astrocytes (20), Schwann and perineural cells (17, 21), and lymphoid follicles (22).
Large alterations of the
6 integrin heavy chain have not
been reported.
Various disease states involving epithelial cells have been associated
with alterations in
6 integrin-containing heterodimers. Mice lacking the
6 integrin completely will develop to
birth but die shortly thereafter because of severe blistering of the skin and other epithelia (23). Alterations in the
6
integrin and/or a deficiency of its pairing subunit,
4
integrin, are associated with pyloric atresia-junctional epidermolysis
bullosa, a human blistering disease of the epithelia (24-29).
Our work investigating a human epithelial cancer indicated a deficiency
of the
6
4 heterodimer pairing during
prostate tumor progression (30, 31) and a persistent expression of the
6
1 integrin (32). Other groups also have
observed the persistent nonpolarized expression of the
6
integrin during human tumor progression in cancers arising within the
breast (33), kidney (34), endometrium (35), and pancreas (36, 37), in
addition to micrometastases from solid epithelial tumors (38).
Isolating the
6 integrin from human prostate cancer
cells using
6-specific monoclonal antibodies retrieved
not only the expected
1 and
4 subunits
but also a predominant protein with an apparent molecular mass
of 70 kDa (7, 8). In this study we show that the protein is a novel and
smaller form of the
6 integrin that is capable of
pairing with either the
1 or
4 integrin subunit, referred to as
6p for the latin word
parvus, meaning small.
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EXPERIMENTAL PROCEDURES |
Cell Lines--
All human cell lines were incubated at 37 °C
in a humidified atmosphere of 95% air and 5% CO2. Cell
lines DU145H, HaCaT, and PC3-N were grown in Iscove's modified
Dulbecco's medium (Life Technologies, Inc.) plus 10% fetal
bovine serum. Cell lines MCF-7, PC3-ATCC, LnCap, and H69 were
grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
plus 10% fetal bovine serum. SW480 cells were grown in super medium
(Dulbecco's modified Eagle's medium plus 5% nonessential amino
acids, 5% L-glutamine, 5% sodium pyruvate, 10% fetal
bovine serum). Normal prostate cells, PrEC, were grown in PrEGM bullet
kit medium (Clonetics, San Diego, CA). The following cell lines were
obtained from the American Type Culture Collection (Manassas, VA):
MCF-7 (human breast tumor), PC3 (human prostate tumor), LnCap (prostate
carcinoma cell line), H69 (human lung carcinoma), and SW480 (human
colon carcinoma). The DU145H cells were isolated by us as described
previously (8) and contain only the
6A splice variant
(30). The PC3-N cells are a variant of PC3 prostate carcinoma cell line
(39). The HaCaT cells (normal immortalized keratinocyte cell line) (40) were obtained from Dr. Norbert E. Fusenig (German Cancer Research Center, University of Heidelberg, Heidelberg, Germany). PrEC (a normal
prostate cell line) was obtained from Clonetics. The calcium-induced terminal differentiation assay, cell culture techniques, and
preparation of calcium medium used for mouse 291, 03C, and 03R cells
have been described previously (41, 42). Cells were maintained in 0.04 mM calcium (low calcium) and switched to medium with 0.14 mM calcium (medium calcium) or 1.4 mM calcium
(high calcium) by 60% confluency. After 24 h treatment, cells
were collected in phosphate-buffered saline, centrifuged, frozen in a
dry ice bath, and kept at
80 °C in a freezer until used.
Antibodies Used in This Study--
Anti-
6
integrin antibodies include and were obtained as follows. GoH3,
a rat IgG2a, was from Accurate Chemicals (Westbury, NY) (43); J1B5, a
rat monoclonal antibody, was a generous gift from Dr. Caroline Damsky,
(University of California, San Francisco, CA) (44); 4F10, a mouse
IgG2b, was from Chemicon (Temcula, CA) (43); BQ16, a mouse IgG1 that
recognizes an external epitope of the
6 integrin, was a
generous gift from Dr. Monica Leibert (Department of Urology,
University of Texas, M.D. Anderson Cancer Center, Houston, TX) (45);
4E9G8, a mouse IgG1 that is specific for the unphosphorylated
6A cytoplasmic tail, was from Immunotech (Marseille,
France) (11, 46); AA6A, a rabbit polyclonal antibody that was raised
and purified by Bethyl Laboratories Inc. (Montgomery, TX) specific for
the last 16 amino acids (CIHAQPSDKERLTSDA) of the human
6A sequence (2) as done previously (47), and A33, a
rabbit polyclonal antibody that was raised against amino acids 1-500
of the
6 integrin (48), were generous gifts from Dr. Arnoud Sonnenberg (The Netherlands Cancer Institute).
Anti-
4 integrin antibodies were obtained as follows.
3E1, a mouse ascites IgG1, was from Life Technologies, Inc. (49);
439.9b, a rat IgG2bK, was from Pharmingen (San Diego, CA) (50); ASC-3,
a mouse IgG1K, was from Chemicon (Temecula, CA) (51); and A9, a mouse
IgG2a, was from Ancell (Bayport, MN) (52). Other anti-integrin
antibodies used include anti-
5 integrin antibody P1D6, a
mouse IgG3 (Life Technologies, Inc.) (53), and anti-
1
integrin P4C10, a mouse ascites IgG1 (Life Technologies, Inc.)
(54).
Surface Biotinylation of Cell Lines--
Previous protocols (55,
56) were slightly modified. Briefly, cells were grown to confluency in
100-mm tissue culture dishes and washed three times with HEPES buffer
(20 mM HEPES, 130 mM NaCl, 5 mM
KCl, 0.8 mM MgCl2, 1.0 mM
CaCl2, pH 7.45). The cells were then incubated with 2 ml of
HEPES buffer supplemented with sulfosuccinimidyl hexanoate-conjugated
biotin (500 µg/ml; Pierce), which is impermeant to cell membranes
(57), to label cell surface proteins for 30 min at 4 °C. The cells
were washed three times and lysed in cold radioimmune precipitation
buffer plus protease inhibitors (phenylmethylsulfonyl fluoride, 2 mM; leupeptin and aprotinin, 1 µg/ml). The lysate was
briefly sonicated on ice before centrifugation at 10,000 rpm for 10 min, and the supernatant was collected for immunoprecipitations.
Immunoprecipitations--
For immunoprecipitations, 200 µg of
total protein lysate was used for each reaction and incubated with 35 µl of protein G-Sepharose and 1 µg of antibody. The final volume of
the lysate was adjusted to 500 µl with radioimmune precipitation
buffer (150 mM NaCl, 50 mM Tris, 5 mM EDTA, 1% (v/v) Triton X-100, 1% (w/v) deoxycholate, 0.1% (w/v) SDS, pH 7.5). The mixture was rotated for 18 h at
4 °C. After incubation, complexes were washed three times with cold radioimmune precipitation buffer and eluted in 2× nonreducing sample
buffer. Samples were boiled for 5 min prior to loading onto a 7.5%
polyacrylamide gel for analysis. The proteins resolved in the gel were
electrotransferred to Millipore (Bedford, MA) Immobilon-P
polyvinylidene fluoride membrane, incubated with either peroxidase-conjugated streptavadin or Western blotting antibodies plus
secondary antibody conjugated to horseradish peroxidase, and visualized
by chemiluminescence (ECL Western blotting detection system; Amersham
Pharmacia Biotech).
Two-dimensional Nonreduced/Reduced Gel
Electrophoreses--
Nonreduced/reduced two-dimensional
electrophoresis was done as described previously (58). The samples were
incubated in 0.625 M Tris-HCl, pH 6.8, 10% glycerol, 10%
SDS, and applied to SDS-polyacrylamide gel electrophoresis (7.5%
acrylamide) without reduction. The excised lanes were incubated in
reducing sample buffer for 15 min and horizontally loaded at the top of
a second dimension slab gel (also 7.5% acrylamide). The proteins were
electrotransferred to polyvinylidene fluoride membrane (Millipore),
incubated with either peroxidase-conjugated streptavadin or
Western blotting primary antibodies followed by secondary antibody
conjugated to horseradish peroxidase, and visualized by
chemiluminescence (ECL Western blotting detection system; Amersham
Pharmacia Biotech).
Amino Acid Sequencing by Matrix-assisted Laser Desorption
Ionization Mass Spectrometry and Liquid Chromatography-Tandem Mass
Spectrometry--
Amino acid sequencing of
6p was
performed using two different analytical core services. For analytical
core service at Deutsches Krebsforschungszentrum (Heidelberg, Germany),
the
6p protein was immunoprecipitated using J1B5, and
the proteins were separated by SDS-polyacrylamide gel electrophoresis
(7.5%, 3 mm). After staining with Coomassie Blue, the
6p bands were excised, cut into small pieces (1 × 1 mm), washed, dehydrated (twice for 30 min with H2O, twice
for 15 min with 50% acetonitrile, and once for 15 min with
acetonitrile), and incubated with 0.5 µg of trypsin in 20 µl of
digest buffer (40 mM NH4HCO3, pH
8.0) at 37 °C for 16 h. The supernatant was subsequently
analyzed by MALDI1 mass
spectrometry (Deutsches Krebsforschungszentrum) using thin film
preparation technique. Aliquots of 0.3 µl of a nitrocellulose containing saturated solution of
-cyano-4-hydroxycinnamic acid in
acetone were deposited onto individual spots on the target. Subsequently, 0.8 µl of 10% formic acid and 0.4 µl of the digest sample were loaded on top of the thin film spots and allowed to dry
slowly at ambient temperature. To remove salts from the digestion buffer, the spots were washed with 5% formic acid and with
H2O. Sequence analysis was performed on a Procise
494 protein sequencer using a standard program supplied by
Applied Biosystems. The FastA data base searching program of Pearson
and Lipman (59) was used for data base searching.
For sequence analysis at the Proteomics Core of the Arizona Cancer
Center and Southwest Environmental Health Sciences Center of the
University of Arizona, the
6p protein was
immunoprecipitated using J1B5, and proteins were separated by
SDS-polyacrylamide gel electrophoresis (7.5%, 3 mm). After staining
with Coomasie Blue, the
6p bands were excised, cut into
small pieces (1 × 1 mm), and subjected to in gel digestion using
trypsin as described previously (60). The extracted peptides following
digestion were analyzed by liquid chromatography-tandem mass
spectrometry using a quadrupole ion trap Finnigan LCQ classic
mass spectrometer equipped with a quartenary pump P4000 HPLC and a
Finnigan electrospray ionization source (ThermoFinnigan, San Jose, CA).
The peptides were eluted from a reverse-phase C18 micro-column (Vydac
250 × 1 mm, Hesperia, CA) with a gradient of 3-95%
acetonitrile in 0.5% formic acid and 0.01% trifluroacetic acid over
150 min at a flow rate of 15 µl/min. Tandem mass spectrometry spectra
of the peptides were analyzed with the SEQUEST program (Turbo Sequest)
to assign peptide sequence to the spectra (61). SEQUEST analyses were performed against the nonredundant data base.
RT-PCR Analysis--
Total cellular RNA was isolated by
guanidium isothiocyanate cell lysis and cesium chloride purification
(62). RNA was quantitated from spectrophotometric absorbance
measurements at 260 nm. First strand cDNA was synthesized in a
30-µl reaction comprised of 1× PCR buffer (10 mM Tris,
pH 8.3, 50 mM KCl, 1.5 mM MgCl2); 1 mM each dATP, dCTP, dGTP, and dTTP; 100 pmol random
hexamer, 20 units RNAsin; 200 units SuperScript reverse transcriptase
II (Life Technologies, Inc.), and 3 µg of total cellular RNA
incubated at 42 °C for 60 min. The reaction was terminated by
incubation at 99 °C for 10 min. Integrin
6-specific
PCR was performed by adding 80 µl of amplification reaction buffer
(1× PCR buffer, 25 pmol of integrin
6-specific primers,
and 2.5 units of Taq DNA polymerase) to the cDNA
reaction, followed by incubation at 94 °C for 5 min and then 40 cycles of 94 °C for 1 min, 60 °C for 3 min, and 72 °C for 10 min, with a final extension at 72 °C for 5 min and a quick chill to
4 °C. The PCR primers were derived from the integrin
6 cDNA sequence reported by Tamura et al.
(2) (GenBankTM accession number X53586); the upstream
primer sequence was from nucleotides 160 to 179, and the downstream
primer was from nucleotides 3404 to 3423. The PCR product identity was
confirmed by diagnostic restriction enzyme digests and size separation
of the products through a 1× TBE, 1.5% agarose gel. The products were
visualized by ethidium bromide staining and UV fluorescence.
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RESULTS |
DU145H Cells Contained a Smaller Form of the
6
Integrin--
Our previous studies showed that anti-
6
antibody GoH3 was able to immunoprecipitate a surface-biotinylated
70-kDa (nonreduced) protein from DU145H cells in addition to the
expected 185-, 140-, and 120-kDa (nonreduced) proteins corresponding to
the
4,
6, and
1 integrins,
respectively (7, 8). In DU145H cells, which only contain the
6A splice variant of
6 integrin (30), this 70-kDa variant was the predominant form of the
6
integrin found on the cell surface.
Five different anti-
6 antibodies immunoprecipitated
6 and its smaller variant,
6p, from
surface-biotinylated DU145H cells (Fig.
1). Four of the antibodies used were
specific for extracellular epitopes of the full-length
6A integrin (GoH3, J1B5, 4F10, and BQ16), and one was
specific for the cytoplasmic tail of the
6A light chain
(AA6A). The integrin
6p was not found to
co-immunoprecipitate upon incubation with an anti-
3
antibody, P1B5 (7), or an anti-
5 antibody, P1D6.

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Fig. 1.
The 6p
integrin was immunoprecipitated from human cells. The DU145H cells
were surface-biotinylated, and the 6 integrin was
retrieved using either the GoH3, J1B5, AA6A, 4F10, or BQ16 antibodies,
specific for human 6 integrin. The 5
integrin was retrieved from the lysate using the P1D6 antibody,
specific for human 5 integrin. The immunoprecipitations
were analyzed using a 7.5% polyacrylamide gel under nonreducing
conditions, and the migration position of the biotinylated integrins
are as indicated.
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The
6p Variant Contained a Light Chain That Was
Identical to That Found in
6 Integrin--
The
full-length
6 integrin consists of two disulfide linked
chains: a heavy chain (110 kDa) and a cytoplasmic light chain (30 kDa)
that are observed upon reduction of the protein samples and analysis by
SDS-polyacrylamide gel electrophoresis. Our data (Fig. 1) indicated
that an anti-
6 integrin antibody specific for the
cytoplasmic tail of
6A recognized
6p and
suggested that the light chain from the
6p variant might
be similar to that in the full-length
6 integrin. To
answer this question, we surface-biotinylated DU145H cells and then
immunoprecipitated the sample using the anti-
6 integrin
antibody, GoH3. The resulting sample was then analyzed using
two-dimensional nonreducing/reducing gel electrophoresis (Fig.
2). The sample was electrophoresed under
nonreducing conditions in the first dimension and then under reducing
conditions for the second dimension. The 160-kDa band (nonreduced)
corresponding to the full-length
6 integrin, contained a
heavy (110 kDa) and light (30 kDa) chain, as described previously,
under the reducing conditions of the second dimension (3). The reduced
1 integrin was identified at 120 kDa. The
6p integrin split into a heavy fragment (43 kDa) and a
light chain (30 kDa). These results indicated that the
6p integrin contained the same light chain as the
full-length
6A integrin but that the heavy chains were
significantly different.

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Fig. 2.
The 6p
integrin contained a light chain identical to the
6 integrin. Surface-biotinylated
proteins from DU145H cells were retrieved by immunoprecipitation using
the GoH3 antibody and were analyzed first by 7.5% polyacrylamide gel
electrophoresis under nonreducing conditions. The resulting lane was
excised from the gel and placed on the top of a second 7.5%
polyacrylamide gel. The position of the migration of the integrins in
the first gel are indicated at the top of the figure.
Electrophoresis was then performed under reducing conditions. The
resulting migration of the heavy chain (HC) and light chain
(LC) and the molecular masses are indicated. The
asterisk indicates a biotinylated protein band that was
variably seen and is of unknown identity.
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The
6p Variant Associated with
1 and
4 Integrins--
The
6 integrin is known
to associate with either the
1 or
4
subunit (10). Next we determined whether
6p would
co-immunoprecipitate with the
4 integrin (Fig.
3A). Human HaCaT cells were
chosen for this experiment because of their abundance of
4 integrin (7). They were surface-biotinylated and
subjected to immunoprecipitation with different anti-
4
integrin antibodies. The
6p variant
co-immunoprecipitated upon incubation with four different
anti-
4 integrin antibodies: A9, 439.9b, ASC3, and 3E1.
Of particular interest was the retrieval of
6p with the
anti-
4 integrin antibody, A9, whose epitope is present
when
6 is coupled to
4 integrin (52).

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Fig. 3.
The 6p
integrin paired with either the 4
or 1 subunits. The HaCaT
cells were surface-biotinylated, and the 4 integrin was
retrieved using either A9, 439.9b, ASC3, or 3E1 antibodies, specific
for human 4 integrin (A). The DU145 cells
were surface-biotinylated, and the 1 integrin was
retrieved using P4C10 antibody, specific for 1 integrin
(B). The immunoprecipitated proteins were analyzed using a
7.5% polyacrylamide gel under nonreducing conditions, and the
migration positions of the biotinylated integrins are as
indicated.
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Next we tested whether the novel 70-kDa (nonreduced) protein could be
recovered by immunoprecipitation with the anti-
1
integrin monoclonal antibody, P4C10 (Fig. 3B). HaCaT cells
were surface-biotinylated and immunoprecipitated with
anti-
6 integrin antibody, J1B5, and used as a standard.
Both DU145 and HaCaT cells were surface-biotinylated and subjected to
immunoprecipitation using P4C10. Interestingly, the 70-kDa (nonreduced)
6p variant co-immunoprecipitated with the
1 integrin in DU145 cells but not in HaCaT cells. The
results indicated that the novel
6p variant paired with
either the
4 or
1 integrin subunits.
Although the
1 integrin was readily present in HaCaT
cells, the
6p integrin did not co-immunoprecipitate with
the anti-
1 integrin antibody, P4C10. This may indicate
that in some cell lines, there is preferential pairing of the
6p integrin subunit with
4.
The
6p Integrin Was Recognized by Light
Chain-specific Anti-
6A Monoclonal Antibodies--
Our
data (Fig. 2) indicated that
6p contained a light chain
identical to that contained in the full-length
6
integrin. Next we tested whether the novel 70-kDa (nonreduced) protein
could be recognized by anti-
6 integrin antibodies via
Western blotting. DU145H cells were biotinylated and immunoprecipitated
with GoH3 for a standard to compare with a Western blot (Fig.
4). DU145H, HaCaT, and H69 cells were
lysed and immunoprecipitated with either anti-
6 integrin
antibodies GoH3 or J1B5 or anti-
1 integrin monoclonal antibody, P4C10. A 70-kDa band that co-migrated with the biotinylated standard was recognized in HaCaT and DU145H cells by Western blot analysis using two different anti-
6A antibodies, AA6A
and 4E9G8, which recognize the cytoplasmic domain of the
6A integrin. Additionally the
6 integrin,
but not the
6p variant, was detected by Western blot
analysis using A33, which is specific for the N-terminal of the
6 integrin. A lung carcinoma cell line, H69, is a cell line that does not contain
6 integrin and was not found
to express
6p.

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Fig. 4.
The 6p
integrin was recognized by antibodies specific for the
6A light chain. The
6 integrin and the 1-containing integrins
were retrieved from the lysates of human DU145H, HaCaT, and H69 cells
by immunoprecipitation with either anti- 6 integrin
antibodies GoH3 or J1B5 or anti- 1 integrin antibody
P4C10. The resulting proteins were analyzed using a 7.5%
polyacrylamide gel under nonreducing conditions followed by Western
blot (WB) analysis using the 6A-specific
antibodies 4E9G8 or AA6A, which are specific for the cytoplasmic
domain, or the anti- 6 integrin antibody A33, which is
specific for the N terminus of 6 integrin. The migration
position of a biotinylated integrin standard from DU145H cells are as
indicated. The samples shown in the middle panel were
electrophoresed on a separate gel, and the molecular mass of the
6A band is indicated relative to the adjacent panels by
a solid bar. The asterisk indicates a
biotinylated protein band that was variably seen and is of unknown
identity.
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The
6p Variant Was Present in Several Different
Epithelial Cancer Cell Lines--
We next determined the presence of
the
6p variant in other tumor or normal cell lines. The
presence of
6 and
6p was initially analyzed by using whole cell lysates (20 µg of total protein) followed by Western blot analysis (data not shown). The results were
tabulated and confirmed by immunoprecipitation with
anti-
6 antibody GoH3 followed by Western blot analysis
using anti-
6A antibody, AA6A (Fig.
5). The
6p variant was
present in several prostate cancer cell lines (DU145H, PC3, and LnCaP)
and a colon cancer cell line (SW480). Additionally,
6p
was present in a normal, immortalized keratinocyte cell line, HaCaT.
The
6p variant was not found in several cell lines
including normal prostate cells, PrEC; a variant of the prostate cell
line PC3, called PC3-N (39); a breast carcinoma cell line, MCF-7; and a
lung carcinoma cell line, H69. Interestingly, the
6p
variant was only observed in cells that expressed the full-length
6 integrin. The
6p variant was not
present in
6-negative cell lines. Two epithelial cell lines, one normal cell line (PrEC) and one cancer cell line (PC3-N), expressed the full-length
6 integrin but not the
6p variant.

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Fig. 5.
The 6p
integrin was present in normal and tumor epithelial cell lines.
The 6-containing integrins were retrieved from the
lysates of normal skin (HaCaT) and prostate epithelial cells (PrEC),
prostate cancer cell lines (PC3, PC3-N, and LnCaP), breast cancer cell
line (MCF-7), colon carcinoma cell line (SW480), and a lung carcinoma
cell line (H69) by immunoprecipitation with the GoH3 antibody. The
immunoprecipitated proteins were analyzed using a 7.5% polyacrylamide
gel under nonreducing conditions. The presence of 6p was
detected by Western blot analysis using the AA6A antibody, specific for
the human 6A light chain.
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The
6p Variant Contained Several Amino Acid
Fragments Identical to the
6 Integrin--
Although
these data showed the presence of the
6A light chain in
the protein, we next determined whether the
6 heavy
chain was present utilizing a direct protein sequencing method. The
6p protein was immunoprecipitated with J1B5 and
electrophoresed. The protein gel was stained with Coomassie Blue, and
the 70-kDa protein was excised and digested with trypsin. Protein
sequences were obtained using either MALDI mass spectrometry (Deutsches Krebsforschungszentrum) or liquid chromatography-tandem mass
spectrometry (Proteomics Core of the Arizona Cancer Center and
Southwest Environmental Health Sciences Center, University of Arizona)
(Fig. 6). Ten noncontinuous amino acid
fragments within the
6p variant were identified that corresponded exactly to predicted trypsin fragments located on exons
13-25 of the published
6 integrin sequence (2). The sequencing data confirmed that both the heavy and light chains of the
6p variant contained identical portions of the
full-length
6 integrin (Fig.
7).

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Fig. 6.
Sequences obtained from the
6p variant corresponded to exons 13-25
of the full-length 6
integrin. The sequences of exons 1-25 of the 6
integrin are indicated by their one-letter amino acid abbreviations.
MALDI mass spectrometry and HPLC coupled to mass spectrometry
identified 10 noncontinuous amino acid fragments from the
6p variant. These corresponded exactly to sequences
contained within 6 exons 13-25 and are indicated by
boxes. Five of nine putative glycosylation sites are
retained within exons 13-25 and are indicated in bold and
underlined type. 10 of 20 cysteine residues (indicated by
closed circles) are retained within exons 13-25.
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Fig. 7.
Schematic of the
6 and
6p integrins. A schematic of the
full-length 6 integrin and the smaller 6p
variant is shown. Repeated domains (shaded rectangles) are
indicated by Roman numerals I-VII. I, 42-79;
II, 113-145; III, 185-217; IV,
256-292; V, 314-352; VI, 375-411;
VII, 430-470. The putative ligand- and cation-binding
domains are contained between repeated domains III and
IV and domains V and VI, respectively.
Exons 1-25 of the 6 integrin sequence are indicated. 10 noncontinuous amino acid fragments obtained from the 6p
integrin corresponded exactly to sequences contained within exons
13-25 of the full-length 6 integrin. The mapped
sequence positions of the two Western blotting anti- 6A
antibodies (AA6A and 4E9G8) that recognize both 6 and
the 6p variant are shown by an asterisk on
the full-length 6 schematic. Conformationally dependent
epitopes for anti- 6 integrin antibodies used for
immunoprecipitation are not indicated on the schematic.
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The
6p Variant Half-life Was Three Times Longer than
6--
Detection of the
6p band that was
smaller than the full-length
6 integrin prompted us to
ask whether this novel variant was a degradation product of the
6 integrin that would be rapidly cleared from the
surface. To answer this question, the surface half-life of both
integrins was determined. Previously we determined that it was possible
to detect the surface half-life of the integrin by biotinylation
strategy (63). The surface proteins of DU145H cells were biotinylated
for 1 h, washed, and placed back in the incubator with medium.
After 24, 48, or 72 h, the integrins were immunoprecipitated using
the GoH3 antibody and analyzed under nonreducing conditions (Fig.
8A). The data indicated that
the
6p form remained on the surface of the DU145H cells
with a half-life of ~72 h, or almost 3 times longer than that of the
full-length
6 integrin (Fig. 8B). The
abundance of
6p was not influenced by exogenous protease
inhibitors (BB94, leupeptin, aprotinin, 30% fetal bovine serum,
ecotin), exogenous proteases (kallikrein), or activators of integrin
function (12-O-tetradecanoylphorbol-13-acetate, 20 mM CaCl2) (data not shown).

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Fig. 8.
The 6p
variant had a longer surface retention time than
6 integrin. A, DU145H
cells were surface-biotinylated and incubated for 24, 48, or 72 h,
followed by lysis and immunoprecipitation with anti- 6
antibody GoH3. The samples were analyzed by a nonreducing 7.5%
polyacrylamide gel, transferred to polyvinylidene fluoride membrane,
reacted with peroxidase-conjugated streptavadin, and visualized by
chemiluminescence. The asterisk indicates a biotinylated
protein band that was variably seen and is of unknown identity.
B, the film was digitized, and the densitometry values were
analyzed for relative degradation rates of 6,
1, and 6p.
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RT-PCR Analysis of the
6 Coding Region Revealed a
Single Product--
RT-PCR was used to determine whether splice
variants of the integrin
6 mRNA were potentially
responsible for the production of the smaller integrin protein. Three
micrograms of total cellular RNA from DU145H cells was
reverse-transcribed into first strand cDNA and then PCR amplified
with primers that essentially bracketed the entire integrin
6 protein coding region (all but the first four codons
were amplified using these primers). The results of this experiment are
shown in Fig. 9. A single PCR product
consistent with a full-length RT-PCR product of 3263 bp was detected;
the splicing out of coding exons would have been detected by the
presence of smaller products in the PCR reaction.

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Fig. 9.
RT-PCR of the
6 integrin coding region revealed a
single PCR product. PCR primers that bracketed the integrin
6 coding region were used to amplify first strand
cDNA generated from cell line DU145H (lane 1). To
confirm the identity of the integrin 6 PCR product,
aliquots were digested with four diagnostic restriction enzymes
(EcoNI, lane 2; EcoRI, lane
3; SmaI, lane 4; and XhoI,
lane 5), size separated on a 1× TBE-1.5% agarose gel, and
visualized by ethidium bromide staining. The molecular mass standard is
EcoRI/HindIII-digested DNA (lane
M).
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To confirm the identity of the integrin
6 PCR product,
diagnostic restriction enzyme digests were performed. Analysis of the
integrin
6 sequence (2) revealed the presence of one
EcoNI site (producing fragments of ~960 and 2300 bp), four
SmaI restriction sites (producing fragments of 105, 150, 350, and 2650 bp), four EcoRI sites (producing fragments of
30, 680, 730, and 1780 bp), and one XhoI site (producing
fragments of 420 and 2840 bp). Aliquots of the integrin
6 PCR product were digested with each of these restriction enzymes, and the results of this experiment are shown in
Fig. 9. Each restriction digest product produced the restriction fragments expected from the integrin
6 PCR product (the
30-bp EcoRI fragment and the 105-bp SmaI fragment
could not be visualized on the gel shown in Fig. 9). Based on these
results, it appears unlikely that the
6p variant is the
result of the splicing out of exons in the known coding region.
Calcium-induced Normal Keratinocyte Differentiation Increased
6p Integrin Protein Levels--
Mouse 291 normal
keratinocyte terminal differentiation can be induced by calcium. O3C
and O3R cells were derived from normal 291 mouse cell strains and are
immortalized, nontumorigenic, and tumorgenic, respectively (64). Both
cell strains are resistant to calcium-induced terminal differentiation.
The presence of
6 and
6p integrins in
normal 291 mouse keratinocytes was determined using whole cell lysates
followed by Western blot analysis using anti-
6 integrin
antibody, AA6A (Fig. 10A).
The results for 291 cells were confirmed by immunoprecipitation with
anti-
6 integrin antibody, GoH3 (data not shown). The
6 and
6p integrin protein bands were
quantitated using Scion Image (65) and graphed (Fig. 10B).
Calcium-induced terminal differentiation increased
6p
integrin protein levels 3-fold in a dose-dependent manner
in 291 nontransformed mouse keratinocytes. The differing steady-state
levels of
6p in proliferating O3C and O3R tumor
cells under the same culture conditions suggested that the
6p integrin variant was responsive to terminal
differentiation and not to calcium itself. Interestingly,
6p integrin levels were decreased in poorly
differentiated squamous cell carcinoma O3R cells relative to initiated
cell O3C precursors and terminally differentiated 291 keratinocytes.

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Fig. 10.
Calcium-induced normal keratinocyte
differentiation increased 6p
levels. The presence of 6 and 6p
integrins was determined in normal 291 mouse keratinocytes,
immortalized O3C nontumorgenic, and O3R tumorgenic derivatives. The
cells were maintained in 0.4 mM calcium (low, lanes
L) and switched to 0.14 mM (medium, lanes
M) or 1.4 mM (high, lanes H) calcium medium
at 60% confluency for 24 h of treatment, then frozen in a dry ice
bath, and kept at 80 °C in a freezer until use. Whole cell lysates
(20 µg) were electrophoresed under nonreducing conditions on a 7.5%
polyacrylamide gel and transferred to polyvinylidene fluoride membrane
followed by Western blot analysis using anti- 6 integrin
antibody, AA6A (A). The 6 and
6p integrin protein bands were scanned and quantitated
using Scion Image and graphed (B).
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DISCUSSION |
Our previous work has shown that the
6 integrin is
associated with an increased invasive potential of human prostate
cancer cells in vitro and the progression of human prostate
carcinoma in human tissue biopsy material. We have found the
6 integrin exists in the classical form (140 kDa,
nonreduced) and in a novel smaller form (70 kDa) referred to here as
6p. The
6p is related to the full-length
6 because it was immunoprecipitated with
anti-
6 integrin antibodies (GoH3, J1B5, AA6A, 4F10, and
BQ16) (Fig. 1). Two-dimensional gel analysis revealed that the light
chain of the
6p integrin was the same size as that found
in the full-length
6 form (Fig. 2). The
6p variant co-immunoprecipitated with both anti-
4 (3E1, A9, 439.9b, and ASC3) and
anti-
1 (P4C10) integrin antibodies (Fig. 3) and was
recognized by two anti-
6 integrin antibodies specific
for the cytoplasmic domain (AA6A and 4E9G8) by Western blot analysis
but not by a polyclonal antibody that was specific for the N-terminal
domain (A33) (Fig. 4). The
6p variant was found in
several different human prostate (DU145H, LnCaP, and PC3) and colon
(SW480) cancer cell lines (Fig. 5). It was not found in several cell
lines including normal prostate cells (PrEC), a breast cancer cell line
(MCF-7), a lung cancer cell line (H69), or a variant of a prostate
carcinoma cell line (PC3-N). MALDI mass spectrometry indicated multiple
amino acid regions in the
6p variant that corresponded
exactly to sequences contained within exons 13-25 of the published
full-length
6 sequence (2) (Figs. 6 and 7).
Calcium-induced terminal differentiation of normal mouse 291 keratinocytes resulted in a 3-fold increase of
6p
protein levels (Fig. 10). It remains to be determined whether a cause
and effect relationship exists between
6p and
differentiation or whether production of the variant simply reflects a
dynamic modulation of cell surface adhesion. Integrin modulation is
known to occur in the differentiation of human keratinocytes (66). Modulation of calcium levels in 291 cell derivatives O3C and O3R cells
that are both resistant to calcium-induced differentiation did not
result in alterations of
6p integrin levels. Together, these data suggest that
6p integrin was responsive to
the dynamic surface modulation induced by terminal differentiation and
that the observed alteration of
6p protein levels was
not solely due to calcium.
The 10 noncontinuous amino acid fragments obtained from the
6p variant corresponded exactly to sequences contained
within exons 13-25 of the full-length
6 integrin (Figs.
6 and 7). No peptide fragments corresponding to exons 1-12 were
obtained using this method, suggesting that the
6p
variant is composed of exons 13-25 of the full-length
6 integrin.
The predicted molecular mass of exons 13-25 is 55 kDa; yet the
6p protein band had an apparent molecular mass of 70 kDa
by gel analysis. This apparent contradiction may be due to a
post-translational modification of the protein. The full-length
6 integrin has a predicted molecular mass of 140 kDa
(2); yet experimentally, the protein band had an apparent molecular
mass of 160 kDa under nonreducing conditions. The variation between
predicted and apparent molecular mass in both proteins is likely due to
the nine glycosylation sites predicted on the
6 protein
and the five that would remain in
6p. Previously,
differences in N-linked glycosylation of the
6 integrin, revealed by endoglycosidase H and
N-glycanase treatments, has accounted for the variation in the apparent
molecular mass of the
6 integrin from platelets and
carcinoma lines (10).
Our data indicated that the novel
6p variant contained a
significant alteration in the heavy chain, which is entirely
extracellular. The current structural model of the
subunit proposes
that the seven N-terminal repeats adopt the fold of a
-propeller
domain (4, 5). These domains contain seven four-stranded
-sheets and
are arranged in a torus around a pseudosymmetric axis. Structural homology studies of enzymes with known
-propeller folds have identified active sites at the top of the
-propeller, typically where adjacent loops run in opposite directions (67-69). Recent studies of the
-propeller domain in integrins have demonstrated that
folds 1 and 3 in the
4 integrin subunit are important
for ligand binding (70), whereas the
5 integrin ligand
binding site is determined by amino acid sequences in repeats 2 and 3 of the N-terminal domain of the
subunit (71). Based on our mass
spectrometry data, which concluded that the
6p variant
contained only exons 13-25, the entire proposed
-propeller domain
would be missing. Thus, it would be likely that the
6p
integrin variant would function as an inactive receptor for cellular
adhesion to the extracellular ligand. The production of this
subunit variant on the cell surface may be a mechanism for regulation
of extracellular adhesion.
Additionally, because integrins are known to be conformationally
dependent molecules with dynamic ligand interactions (72), alteration
of the extracellular portion of the molecule could likely influence
intracellular signaling (73). The integrin
subunit cytoplasmic
domains have been shown to be important for a diverse number of
functions including adhesion, motility, internalization,
differentiation, and cytoskeletal organization (74-79). Recently, the
role of the
6A cytoplasmic domain was examined in
myoblasts and found to inhibit proliferation and promote
differentiation. Interestingly, the cytoplasmic tail alone suppressed
signaling through the focal adhesion kinase and mitogen-activated
protein kinase pathways (80). A previous report indicated that
post-translational processing of the
4
1
integrin can occur in leukocytes (81), but to our knowledge, this is
the first description of a naturally occurring variation of this size
in the extracellular domain of the
6 integrin subunit.
Interestingly, our data indicated that the altered extracellular region
of the
6p variant did not affect its ability to remain paired with either
4 or
1 integrin
subunits. The
6p variant was retrieved by
immunoprecipitation using the anti-
4 integrin monoclonal
antibody, A9, whose epitope is present when
6 is coupled to the
4 subunit (52). This finding suggests that the
6p subunit is able to heterodimerize with the
4 subunit in the same manner as the full-length
6 integrin. It remains to be determined whether the
6p
4 integrin is functional with its cytoplasmic
binding partners. It is also noteworthy that
6p
co-immunoprecipitated with
1 integrin in DU145H cells
but not in HaCaT cells, despite abundant levels of
1
integrin in the HaCaT cells (Fig. 3B). This finding may
indicate a preferential pairing of
6p to
4 in some cell lines.
Previous studies suggested that integrins and TM4 tetraspan proteins
could interact with one another to modulate integrin signaling and
adhesion (82, 83). Recently, it has been demonstrated that two members
of this family, CD9 and CD81 can interact with the extracellular domain
of the
6 integrin (84). It would be of interest to know
whether the variant
6p retains the ability to bind to
either of these tetraspan proteins.
In regard to the origin of the
6p variant, our data
suggest several possibilities. Information obtained from cell surface retention half-life studies revealed that
6p (70 kDa)
was almost three times more stable than that of the full-length
6
form (Fig. 8). From this data, we concluded the
6p
protein was not a degradation product of the full-length
6 integrin because the protein was not preferentially
cleared from the surface as might be expected for a protein targeted
for degradation. The
6p protein was not generated after
cell lysis, because multiple antiproteases and short
immunoprecipitation times were unable to alter the presence of this
variant. Although some integrins are highly susceptible to proteolytic
processing, i.e. the
4 integrin (85), the
fully processed
6 integrin has not yet been reported to
be enzymatically cleaved by any enzymes in vivo. We were
unable to induce proteolytic cleavage of the
6 integrin
in vivo in our previous studies (85). Collectively, these
findings argue against
6p being a degradation product;
however, they do not provide information as to whether
6p was generated through a post-transcriptional
processing event or alternative splicing of
6 message.
Our data does not suggest that
6p originated from an
alternative splicing event, because analysis by RT-PCR revealed that only one transcript for
6 was present within the known
coding region (Fig. 9). Moreover, it has not previously been
demonstrated in humans that alternative splicing plays a role in the
regulation of the extracellular domain of integrins (6). Several
integrins including
6 have been shown to have isoforms
of the cytoplasmic domain generated by alternative splicing (2, 9, 47).
Our data demonstrated a significant variation (a 70-kDa change) in the
extracellular heavy chain of the
6p integrin (Fig. 2).
This large extracellular variation has not been described previously for other integrins.
Taken together, our data suggest that a post-transcriptional event is
responsible for the generation of
6p. The
6 integrin subunit, in addition to other
subunits,
normally undergoes endoproteolytic processing close to the C terminus
after synthesis, resulting in the formation of a light and heavy chain
(86). A previous report demonstrated that defective
post-transcriptional processing of the pre-
6 transcript
in carcinoma cells lead to loss of normal cleavage and a resulting
larger 150-kDa single protein (87). Examples of normal
post-transcriptional processing have been described in yeast via
translational introns that can give rise to two different sized
proteins from a single mRNA transcript (88). Alternatively, ribosomal scanning past the conventional initiation codon has been
described for major histocompatability class I molecules. In
this process, the ribosome initiates translation further downstream (89). We note with interest that 12 alternative initiation codons are
predicted within the
6 gene and one (position 1833)
precedes exon 13. The mechanism for generating the
6p
heavy chain and the functional role of the variant in adhesion and
signaling processes remain to be determined.