From the
Integrin
Mono-ADP-ribosylation is a post-translational modification of
proteins, in which an ADP-ribose moiety is attached covalently to one
of a number of amino acids
(1) . The best characterized
mono-ADP-ribosylation reactions are those catalyzed by bacterial toxins
(2) , but similar reactions have been also detected in many
eukaryotic organisms
(3, 4) . In skeletal muscle, an
enzyme catalyzing transfer of ADP-ribose from NAD to arginine in
proteins was recently purified
(5, 6) and cloned
(6) . The enzyme was found to be a highly conserved
(7) ,
glycosylphosphatidylinositol (GPI)
Integrins are a family of heterodimeric
integral membrane proteins that mediate the interaction of cells with
the extracellular matrix (ECM). Integrin binding to ECM proteins
regulates cell shape, migration, growth, and differentiation
(9, 10) . In many cases, integrins transduce information
from ECM to trigger intracellular signaling pathways
(11, 12, 13) . The specificity of integrin
interaction with specific components of ECM is conferred by particular
combinations of
Integrin
It has been proposed that
ADP-ribosyltransferase may be coupled with an ADP-ribosylarginine
hydrolase in an ADP-ribosylation cycle
(3) . Since the
hydrolases are ubiquitously expressed and cytosolic
(20) , the
role of these enzymes in reversing the action of the GPI-linked
transferase is unclear. It seemed feasible that cell surface
ADP-ribosylated proteins might undergo a processing that is different
from intracellular pathways. In this report, we addressed the question
of processing of ADP-ribosylated integrin
Incubation of intact differentiated mouse skeletal muscle
C2C12 myoblasts (myotubes) with
[ adenylate-
To determine whether the 102- and 105-kDa
ADP-ribosylated proteins represented different forms of integrin
It is not clear why the mobilities of
integrin
The ratio of
radioactivity associated with the 105- and 102-kDa bands after labeling
of cells with 75 µ
M [
Following incubation of
cells with 5 µ
M [
Radioactivity associated with integrin
The removal
of [
In the presence of external NAD, integrin
Extracellular
ADP-ribose, free or linked to integrin
(Phospho)ribosylarginine is much more stable than
ADP-ribosylarginine in the extracellular compartment. 1 h after
termination of the labeling,
If ADP-ribosylation of integrin
ADP-ribosylation of integrin
C2C12
myotubes (day 10 after plating) were incubated for 15 min in the
presence of 75 µ
M [ adenylate-
We thank Dr. Myron Jacobson for supplying
[ ribose-U-
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
7 is a major substrate in skeletal muscle cells
for the cell surface, glycosylphosphatidylinositol-anchored,
arginine-specific ADP-ribosyltransferase. Since ADP-ribosylarginine
hydrolase, the enzyme responsible for cleavage of the
ADP-ribosylarginine bond and a component with the transferase of a
putative ADP-ribosylation cycle, is cytosolic, the processing of
ADP-ribosylated integrin
7 was investigated. Following incubation
of differentiated mouse C2C12 myoblasts with
[ adenylate-
P]NAD and analysis by
SDS-polyacrylamide gel electrophoresis under reducing conditions, two
[
P]ADP-ribosylated forms of integrin
7 were
re-solved. By pulse-chase and purification of the radio-labeled
proteins on a laminin affinity column, it was demonstrated that a
105-kDa ADP-ribosylated form originated from a mono-ADP-ribosylated
102-kDa form and represented integrin
7 modified at more than one
site. The additional site(s) of modification, utilized at higher NAD
concentrations, were located in the 63-kDa N-terminal segment of
integrin
7. Both [
P]ADP-ribosylated
integrins were loosely associated with the cytoskeleton, bound to
laminin affinity columns, and immunoprecipitated with antibodies to
integrin
1.
P label was rapidly removed from
[
P]ADP-ribosylated integrin
7 at either
site of modification, a process inhibited by free ADP-ribose or
p-nitrophenylthymidine-5`-monophosphate, an alternative
substrate of 5`-nucleotide phosphodiesterase. The processed integrin
7 was unavailable for subsequent ADP-ribosylation, although the
amount of surface integrin
7 remained constant. During the
processing, no loss of label was observed from integrin
7
radiolabeled with [
C]NAD, containing
C in the nicotinamide proximal ribose, consistent with
degradation of the ADP-ribose moiety by a cell surface 5`-nucleotide
phosphodiesterase. Thus, cell surface ADP-ribosylation, in contrast to
intracellular ADP-ribosylation, is not readily reversed by
ADP-ribosylarginine hydrolase and seems to operate outside the
postulated ADP-ribosylation cycle.
(
)
-anchored
protein
(6, 7) located on the cell surface
(7, 8) .
and
subunits
(9) .
7 was a major cell surface substrate for an arginine-specific
ADP-ribosyltransferase in mouse skeletal muscle cells
(8) . The
dimer of integrins
7 and
1 is a laminin-binding protein
(14, 15, 16) involved in cell adhesion and
communication between myoblasts and the extracellular matrix. Integrin
7 is alternatively spliced in both its cytoplasmic
(17, 18, 19) and extracellular domains
(17) . This structural diversity may reflect complexity of
adhesion-mediated signaling pathways, with ADP-ribosylation being one
of the mechanisms regulating these events.
7 and demonstrate that,
in skeletal muscle myotubes, 5`-nucleotide phosphodiesterase, and not
ADP-ribosylarginine hydrolase, is involved in the processing
ADP-ribosylated proteins at the cell surface.
Cell Cultures and Cell Treatment
C2C12
myoblasts (ATCC) were grown on plastic dishes (Falcon) in
Dulbecco's modified Eagle's medium with 10% fetal bovine
serum at 37 °C under a humidified atmosphere with 5%
CO. Cell fusion and formation of myotubes were observed
usually 5 days after plating. Cells were subcultured (
2
10
cells/cm
) on day 3 before the onset of
differentiation. ADP-ribosylation in intact cells was carried out at
room temperature in DPBS (pH 7.2, with 0.9 m
M CaCl
and 0.5 m
M MgCl
) as described earlier
(8) , with [ adenylate-
P]NAD
(DuPont NEN) and ADP-ribose or
p-nitrophenylthymidine-5`-phosphate, as indicated in the
figure legends. Reaction was usually terminated by removing the
labeling medium and adding SDS-PAGE gel loading buffer directly to
plates. Lysed cells were then scraped, boiled for 10 min, and passed
several times through a 25-gauge needle; proteins were resolved by
SDS-PAGE in 5% gels (with the exception of the partial trypsinization
experiment, in which proteins were separated in 8% gels). To study the
processing of ADP-ribosylated integrin
7,
[ ribose-U-
C]NAD, with the radiolabel
present in the nicotinamide-proximal ribose (225 mCi/mmol), was
utilized in experiments similar to those described above. The
[
C]NAD, manufactured by Amersham, was obtained
from Dr. Myron Jacobson (University of Kentucky, College of Pharmacy,
Lexington, KY) and HPLC purified on Zorbax SAX column prior to use.
Extraction of Membrane Proteins with Triton
X-100
Following labeling with
[P]NAD, cells were incubated with ice-cold 20
m
M Tris-Cl (pH 7.5), 150 m
M NaCl, 1 m
M N-ethylmaleimide, 1 m
M MnCl
, 1
m
M phenylmethylsulfonyl fluoride, 1 m
M ADP-ribose,
and 0.5% (v/v) Triton X-100 (0.1 ml of buffer/cm
). For
purification of integrin
7, proteins were extracted with buffer
containing 50 m
M octyl glucoside instead of Triton X-100.
Laminin affinity column was prepared, and integrin
7 purification
was performed as described
(8) .
Immunoprecipitation of Integrin
C2C12 cells were solubilized in 20 m
M
Tris-Cl (pH 7.5), 150 m
M NaCl, 1 m
M MnCl1
,
1 m
M phenylmethylsulfonyl fluoride, 1 m
M ADP-ribose,
and 1% (v/v) Triton X-100 (0.1 ml of buffer/cm
). After
centrifugation (10 min, 14,000
g), 1 ml of the
supernatant was incubated on ice for 2 h with 20 µl of polyclonal
anti-integrin
1 antibody (Chemicon International) and then for 1 h
with 50 µl of protein G-Sepharose (Pharmacia Biotech Inc., 50%
slurry). Protein G-Sepharose beads were sedimented at 5,000
g for 5 min, washed five times with 1 ml of the solubilization
buffer, and boiled for 10 min with 50 µl of SDS-PAGE gel loading
buffer.
Cell Surface Biotinylation
Cells were
washed three times with DPBS, incubated for 30 min on ice with DPBS,
0.9 m
M CaCl, 0.5 m
M MgCl
, and
sulfo-NHS-biotin (0.5 mg/ml, 0.5 ml/cm
, Pierce). After
labeling, cells were washed three times with Dulbecco's modified
Eagle's medium, then three times with DPBS, followed by
extraction with Triton X-100-containing buffer and immunoprecipitation
with anti-integrin
1 antibody. The antibody was specific for the
intracellular domain of integrin
1; therefore, the recognition of
integrin
1 by the antibody was not affected by cell surface
biotinylation. Following separation of the immunoprecipitated proteins
by SDS-PAGE and transfer to the nitrocellulose membrane, biotinylated
proteins were detected with streptavidin-horseradish peroxidase
conjugate (Amersham, 1:2000 dilution) and enhanced chemiluminescence.
Assaying the Extracellular 5`-Nucleotide
Phosphodiesterase Activity
C2C12 myotubes were incubated at
20 °C in DPBS with 1 m
M MgCl, 1 m
M
CaCl
, and 0.5 m
M NP-TMP (3 ml/100-mm plate). The
formation of p-nitrophenol was determined by measuring the
absorbance at 400 nm of the samples of the extracellular buffer
(21) , using the extinction coefficient of 16,000
cm
m
M
. To examine
whether the phosphodiesterase is a GPI-anchored protein, cells were
incubated for 1 h in DPBS with PI-PLC (from Bacillus
thuringiensis, 0.5 unit/ml, 2 ml total volume), the external
buffer was removed, and the enzyme activity was measured in the buffer
and in the cells on plates separately.
HPLC Chromatography
After incubation of
[ adenine-U-C]ADP-ribose with intact
C2C12 cells, the reaction products were separated by chromatography on
Zorbax SAX HPLC column. The column was eluted with 50 m
M
sodium phosphate, pH 4.5 (flow rate, 1 ml/min).
[ adenine-U-
C]ADP-ribose was generated
from [ adenine-U-
C]NAD (Amersham, 270
mCi/mmol) with NADase (partially purified from rat brain and obtained
from Dr. Lee McDonald) and HPLC purified prior to use.
Quantification of the Amount of Radioactivity or
Protein Associated with the 102- and 105-kDa Forms of Integrin
Proteins in SDS-polyacrylamide gels were stained
with Coomassie Blue; gels were dried, and radioactivity was quantified
with PhosphorImager (Molecular Dynamics). After purification of
integrin 7
7 on a laminin column, proteins were analyzed by
SDS-PAGE; gels were silver stained, and intensity of the staining was
quantified by densitometry (Molecular Dynamics). The intensity profile
(radioactivity or protein staining) along a gel lane was measured,
using ImageQuant software (Molecular Dynamics). Each lane was scanned
at least three times. Intensity curves were deconvoluted into two
separate peaks using Origin software (MicroCal), assuming a Gaussian
shape for each peak. The area under each peak was considered to be a
measure of radioactivity or protein content associated with the
respective band on the gel. When absolute amounts of radioactivity were
determined ( e.g. Fig. 1
, B and C), the
gel was sliced without fixation of proteins; slices were incubated for
12 h in 1
M KOH at 60 °C, then neutralized to pH
7.0,
and radioactivity was measured by liquid scintillation counting.
Figure 1:
Time course of ADP-ribosylation of
integrin 7 in intact C2C12 myotubes. A, intact,
differentiated C2C12 cells (day 10 after plating) were incubated with 1
m
M ADP-ribose and the following concentrations of
[ adenylate-
P]NAD: 1 µ
M (20
Ci/mmol, a) 5 µ
M (10 Ci/mmol, b), or 75
µ
M (2.7 Ci/mmol, c). After the indicated time,
100-µg samples of total cellular protein were analyzed by SDS-PAGE
and autoradiography. The amount of radioactivity in each lane was quantified with a PhosphorImager, and the intensity profiles
obtained for the regions of 95-110 kDa were resolved into two
bands. The extent of ADP-ribosylation of the 102-kDa ( B) or
the 105-kDa ( C) form of integrin
7 was evaluated (
,
1 µ
M NAD;
, 5 µ
M NAD;
, 75
µ
M NAD), as described under ``Materials and
Methods.'' Data in B and C are the means
± S.E. from four quantifications. Positions of standard proteins
(kDa) are indicated on the left.
P]NAD resulted in
[
P]ADP-ribosylation of the extracellular domain
of integrin
7
(8) . Earlier, on SDS-PAGE under reducing
conditions, the radiolabeled fragment of integrin
7 behaved as a
protein of 97 kDa
(8) . Here, following incubation of C2C12
myotubes with [ adenylate-
P]NAD,
proteins were analyzed by SDS-PAGE in 5% gels, which gave optimal
resolution of high molecular weight species. In the presence of a
reducing agent, two radiolabeled proteins of
102 and
105 kDa
were detected. These had been unresolved previously, and both ascribed
to the 97-kDa component. The 105-kDa band was very weak when cells were
incubated with 1 or 5 µ
M [
P]NAD
(Fig. 1 A, a and b) and much more pronounced
when the labeling was performed with 75 µ
M
[
P]NAD (Fig. 1 A, c).
Labeling of the 102-kDa protein occurred first, with the 105-kDa
species being modified later in the incubation (Fig. 1, B and C).
7, which were modified independently, or whether the 105-kDa band
originated from the 102-kDa species, which in the presence of a higher
NAD concentration was modified to a greater extent and had a lower
mobility, a pulse-chase experiment was performed. C2C12 myotubes were
first incubated in the presence of 5 µ
M
[
P]NAD, which was followed by a chase in the
presence of 75 µ
M unlabeled NAD. The intensity of the
102-kDa band, radiolabeled during the pulse phase, was gradually
diminished in parallel with the appearance of the 105-kDa radiolabeled
band (Fig. 2, A and B). The shift in the mobility was
completely blocked by incubation of cells in the presence of PI-PLC
between pulse and chase phases (Fig. 2 C). It was
demonstrated earlier that PI-PLC releases GPI-anchored
ADP-ribosyltransferase from intact cells
(7, 8) .
Indeed, as shown in the control experiment in Fig. 2 C,
incubation of C2C12 cells with PI-PLC prior to the incubation with
[
P]NAD prevented any subsequent modification of
the 102-kDa protein. Replacement of NAD with the same concentration of
NADP or NADH during the chase phase did not lead to the mobility shift
of the radiolabeled 102-kDa protein (Fig. 2 D); a slight
mobility shift observed in the presence of NADH can probably be
attributed to its partial oxidation to NAD. Thus, the two radioactive
proteins detected by SDS-PAGE corresponding to 102 and 105 kDa
represent the integrin
7 molecule, modified by the GPI-anchored
ADP-ribosyltransferase, most probably with a different number of sites
ADP-ribosylated.
Figure 2:
Pulse-chase labeling of integrin 7 in
intact C2C12 cells. A, C2C12 myotubes (day 10 after plating)
were incubated for 30 min with 5 µ
M
[ adenylate-
P]NAD and 1 m
M
ADP-ribose and then washed three times with DPBS, incubated for
indicated amounts of time in the presence of 75 µ
M
unlabeled NAD and 1 m
M ADP-ribose, and analyzed by SDS-PAGE
and autoradiography. B, the amount of radioactivity associated
with 102-kDa (
) and 105-kDa (
) forms of integrin
7 was
quantified after different amounts of time of chase with unlabeled NAD.
Data are the means ± S.E. from three quantifications.
C, effect of the incubation of C2C12 cells with PI-PLC on the
change of the mobility of the 102-kDa form of integrin
7 during
the chase with 75 µ
M NAD. The experiment consisted of
three incubations: I, for 30 min; II, for 60 min; and III, for 30 min.
ADP-ribose (1 m
M) was included in the medium in all three
phases of the experiment, with or without NAD or PI-PLC (from B.
thuringiensis, 0.5 unit/ml), as listed below each
lane. In a control experiment ( last lane),
incubation of cells with PI-PLC prior to [
P]NAD
prevented any subsequent labeling of the 102-kDa protein, consistent
with complete removal of the ADP-ribosyltransferase from the membrane.
D, cells were labeled for 30 min with 5 µ
M
[ adenylate-
P]NAD in the presence of 1
m
M ADP-ribose and then incubated for 60 min with 1 m
M
ADP-ribose and 75 µ
M unlabeled NAD, NADP, or NADH.
Alternatively, after the labeling, cells were incubated with ADP-ribose
only ( C, control).
Integrin 7, ADP-ribosylated to different
extents, was purified on a laminin affinity column. C2C12 cells were
incubated with 0, 5, or 75 µ
M
[
P]NAD prior to extraction with detergent.
Following laminin chromatography, the material eluted from the column
was analyzed by SDS-PAGE. A silver-stained gel revealed the presence of
the 102-kDa protein in the absence of ADP-ribosylation or after
ADP-ribosylation in the presence of 5 µ
M NAD. After
incubation of cells with 75 µ
M NAD, strong 102-kDa and
weak 105-kDa protein bands were detected (Fig. 3 A). These data
are consistent with the generation of the 105-kDa form from the 102-kDa
species by ADP-ribosylation.
7, which was not ADP-ribosylated and which was modified
in the presence of 5 µ
M NAD, were the same and why
ADP-ribosylation at higher NAD concentrations induced the large
mobility shift (Fig. 3 A). The apparent difference in size
between the two radiolabeled proteins generated in the presence of 75
µ
M NAD is too much to be accounted for by one ADP-ribose,
which has a molecular weight of
550. Nevertheless, the 105-kDa
species seems to be the first larger discrete radiolabeled form,
observed at higher NAD concentrations, after the 102-kDa one. With
phosphorylated proteins, it has been documented that addition of a
single, small, negatively charged group to a protein can significantly
decrease its mobility in SDS-polyacrylamide gels
(22) , and a
similar effect may be produced by ADP-ribosylation of integrin
7
at a site being modified at higher NAD concentration.
P]NAD and
purification on a laminin column was significantly higher than the
protein ratio for those two bands (Fig. 3, A and B,
and Table I), consistent with a higher extent of labeling of the
105-kDa protein than of the 102-kDa one. If the 105-kDa protein
represents integrin
7 modified with two ADP-ribose moieties and
the 102-kDa protein corresponds to both integrin
7 modified with
one ADP-ribose and to unmodified protein, the relative amounts of each
of the three forms could be estimated; e.g. after 15 min of
incubation of cells with 75 µ
M
[
P]NAD, integrin
7 appears to be a
heterogenous population of molecules, modified with two ADP-ribose
moieties (
16%), with one ADP-ribose (
59%) and one without
modification (
25%) ().
P]NAD and partial
tryptic digestion, 39-kDa and 79-kDa radiolabeled proteins were
observed after SDS-PAGE under reducing and non-reducing conditions,
respectively (Fig. 4, A and B; see also Ref. 8). The
site of ADP-ribosylation was localized to the 39-kDa segment in the
extracellular domain between amino acids 575 and 886 in the integrin
7 sequence
(8, 16) . Partial trypsin digestion
after incubation of cells with 75 µ
M
[
P]NAD produced an additional radiolabeled
66-kDa fragment under reducing conditions (Fig. 4 A)
and
70 kDa under non-reducing conditions (Fig. 4 B).
Because of the similarity of sizes of this fragment under reducing and
non-reducing conditions, we infer that it may correspond to the 63-kDa
N-terminal domain of integrin
7
(16, 17) . This
fragment is not radiolabeled in the pulse-chase experiment
(Fig. 4 A), consistent with the initial ADP-ribosylation
occurring only on the 39-kDa fragment.
Figure 4:
Partial trypsin digestion of
ADP-ribosylated integrin 7. Intact myotubes were incubated for 30
min with 5 µ
M ( lanes 1, 2,
5, and 6) or 75 µ
M
[ adenylate-
P]NAD ( lanes 3 and 4) and 1 m
M ADP-ribose, treated with 0.02%
(w/v) trypsin for 5 min ( lanes 2, 4, and
6), and analyzed by SDS-PAGE in 8% gels under reducing
( A) or non-reducing conditions ( B). In one case
( A, lanes 5 and 6), the labeling
was followed by 1 h in the presence of 75 µ
M unlabeled NAD
and 1 m
M ADP-ribose prior to the trypsinization. Positions of
standard proteins (kDa) are indicated on the left. Positions
of the radiolabeled fragment produced by trypsinization after labeling
of cells with 75 µ
M [
P]NAD are
indicated by arrows. Autoradiograms are
shown.
The two ADP-ribosylated forms
of integrin 7 were readily extracted from cells with Triton X-100
(Fig. 5 A), consistent with their loose interaction with the
cytoskeleton. The relative amounts of radioactivity associated with the
102- and 105-kDa species before and after purification on laminin
affinity column were similar (Fig. 5 B), confirming that
ADP-ribosylation of the 63-kDa, presumably N-terminal, segment of
integrin
7 did not prevent its binding to immobilized laminin.
Both ADP-ribosylated forms of
7 protein were immunoprecipitated
with an antibody against integrin
1 (Fig. 5 C),
demonstrating that regardless of different extents of ADP-ribosylation,
their association with the
1 subunit was similar.
Figure 5:
Comparison of the properties of the 102-
and 105-kDa ADP-ribosylated forms of integrin 7. C2C12 myotubes
were incubated with 75 µ
M
[ adenylate-
P]NAD and 1 m
M
ADP-ribose for 30 min ( lanes 1-5 and
9-11) or 15 min ( lanes 6-8).
Proteins extracted from cells with 1% Triton X-100 during four
consecutive 2-min intervals ( lanes 1-4) or not
extracted ( lane 5) were subjected to SDS-PAGE and
autoradiography. Equal fractions of the material obtained at each step
were analyzed. Proteins extracted with octyl glucoside were applied to
a laminin affinity column, and samples (3% of the total volume) of cell
extract ( lane 6), flow-through ( lane 7), or eluate from the column ( lane 8)
were analyzed by SDS-PAGE and autoradiography. After solubilization
with Triton X-100, immunoprecipitation with anti-
1 antibody was
performed. 20 µl of cell extract ( lane 9),
material immunoprecipitated from 1.5 ml of the extract ( lane 10), or control precipitate (no primary antibody added,
lane 11) were subjected to SDS-PAGE and
autoradiography.
To observe
significant incorporation of [P]ADP-ribose into
integrin
7 during incubation of C2C12 cells with
[ adenylate-
P]NAD, a relatively high
concentration (
1 m
M) of ADP-ribose had to be included in
the incubation medium
(8) . Since ADP-ribose does not have any
direct stimulatory effect on the ADP-ribosyltransferase, this suggested
that enzymatic degradation of the product might be inhibited by free
ADP-ribose. ADP-ribosylarginine hydrolase, which has been identified
recently in a variety of mammalian tissues
(20) , is a cytosolic
enzyme and therefore unlikely to be involved in the removal of
ADP-ribose from proteins at the cell surface. Degradation of
ADP-ribosylarginine could, however, be stepwise, with hydrolysis of the
phosphodiester bond in ADP-ribose as the first step. Indeed,
[ adenine-U-
C]ADP-ribose, incubated with
intact C2C12 cells, was hydrolyzed to [
C]AMP,
which was then converted to [
C]adenosine (Fig.
6 A). In addition, extracellular phosphodiesterase activity of
differentiated C2C12 cells (day 12 after plating) was estimated as 1.27
± 0.11 nmol
min
mg
protein
( n = 3), using NP-TMP as a
substrate
(21) . Accordingly, unlabeled ADP-ribose or NP-TMP
inhibited degradation of
[ adenine-U-
C]ADP-ribose (Fig.
6 A).
7 was
almost completely lost during 1 h in the absence of ADP-ribose or
NP-TMP after termination of the labeling (Fig. 6 B) but
was well preserved in their presence (Fig. 6 B). The
presence of ADP-ribose
(8) or NP-TMP in addition to
[ adenylate-
P]NAD was required during
the labeling reaction to observe significant
[
P]ADP-ribosylation of integrin
7 (Fig.
6 C). In this case, ADP-ribose and NP-TMP prevented the
hydrolysis of both ADP-ribose linked to the integrin and
[
P]NAD, which was also a good phosphodiesterase
substrate and was rapidly degraded when present in micromolar
concentrations, as used for ADP-ribosylation.
Figure 6:
Effect of ADP-ribose and NP-TMP on the
degradation of ADP-ribose in intact C2C12 cells. A,
[ adenine-U-C]ADP-ribose (20
µ
M, 3000 cpm/nmol, 5 nmol/cm
) was incubated
with intact C2C12 myotubes in DPBS, and 200-µl samples of the
incubation medium were analyzed by HPLC on SAX column before (
)
or after 1 h of incubation (
,
, and ▾). Reaction was
carried without additions (
,
) or with 1 m
M
unlabeled ADP-ribose (
) or 1 m
M NP-TMP (▾).
Positions of adenosine ( Ade), AMP, and ADP-ribose
( ADPR) are indicated. B, cells were incubated for 30
min with 5 µ
M
[ adenylate-
P]NAD in the presence of 1
m
M ADP-ribose, which was followed by a 1-h incubation period
in PBS only or with 1 m
M ADP-ribose or NP-TMP, as indicated.
C, C2C12 myotubes were labeled with 5 µ
M
[ adenylate-
P]NAD without or with
ADP-ribose or NP-TMP. In B and C, autoradiograms of
the gels are shown.
Cell
surface-associated phosphodiesterase has been reported to be a
GPI-anchored protein in several tissues
(23) . After incubation
of C2C12 with PI-PLC, 92.9 ± 5.7% ( n = 3) of the
phosphodiesterase activity, assayed with NP-TMP as a substrate,
remained associated with the cells. In control cells, with no PI-PLC
treatment, 94.5 ± 4.7% ( n = 3) of the activity
was associated with the cell surface. This argues either against GPI
anchoring of the muscle cell surface phosphodiesterase or for the
presence of a PI-PLC-resistant GPI-anchor
(24) .
P]AMP from
[
P]ADP-ribosylated integrin
7, catalyzed by
the cell surface phosphodiesterase, would result in formation of a
phosphoribosylarginine residue in the integrin structure. The lifetime
of the phosphoribosylarginine or ribosylarginine, after
dephosphorylation by cell surface phosphatases
(25) , was probed
in ``relabeling'' experiments. C2C12 cells were first
incubated for 30 min with different concentrations of unlabeled NAD and
1 m
M ADP-ribose. Then, the incubation was continued for 1 h in
PBS with ADP-ribose only, followed by radiolabeling with 5
µ
M [
P]NAD and 1 m
M
ADP-ribose (Fig. 7 A). In this case,
[
P]ADP-ribosylation of integrin
7 was
significantly diminished after prior incubation of cells with 5 or 75
µ
M NAD. ADP-ribose, transferred to the protein during the
first phase of the experiment, remained attached during the second
phase (no NAD, high ADP-ribose concentration, see Fig. 6 B) and
obviously blocked the modification with
[
P]ADP-ribose during the third phase.
Importantly, when ADP-ribose was omitted during the second incubation,
allowing phosphodiesterase to degrade ADP-ribose linked to integrin
7 (see Fig. 6 B), radiolabeling of the integrin was
also diminished (Fig. 7 B). Surface biotinylation of
C2C12 cells and immunoprecipitation with anti-
1 integrin antibody
revealed a similar level of biotinylation of 102-105-kDa proteins
in each case (Fig. 7, C and D). The
biotinylated 102- and 105-kDa protein bands colocalized with the
radiolabeled bands observed after
[
P]ADP-ribosylation and immunoprecipitation with
the same antibody (see Fig. 5), and therefore, they were
attributed to integrin
7. Thus, ADP-ribosylation or
(phospho)ribosylation of integrin
7 after the second phase of the
experiment shown in Fig. 7did not have a detectable effect on
its degradation or internalization. Rather, the unavailability of
integrin
7 for subsequent
[
P]ADP-ribosylation suggested that the
ADP-ribosylation site in integrin
7 was still blocked.
Figure 7:
Unavailability of modified integrin 7
for subsequent [
P]ADP-ribosylation.
Differentiated C2C12 cells were incubated for 30 min with 1 m
M
ADP-ribose ( ADPR) with 0, 5, or 75 µ
M unlabeled
NAD, as indicated, then for 1 h without NAD in the presence of 1
m
M ADP-ribose ( A, C) or in the absence of
ADP-ribose ( B, D), which was followed by 30 min of
labeling with 5 µ
M
[ adenylate-
P]NAD and 1 m
M
ADP-ribose ( A, B) or cell surface biotinylation and
immunoprecipitation with anti-integrin
1 antibody ( C,
D). A and B,[
P]ADP-ribosylation of integrin
7 was
visualized after SDS-PAGE and autoradiography. C and
D,biotinylated proteins separated by SDS-PAGE were
detected as described under ``Materials and Methods.'' The
120-kDa, most abundant protein corresponds to integrin
1, the
102- and 105-kDa bands represent integrin
7, and
160- and
200-kDa proteins may correspond to other integrin
subunits,
which coimmunoprecipitated with integrin
1.
To
demonstrate directly the presence of a ribose moiety at the site of
modification in integrin 7, after removal of
P label,
cells were labeled in parallel with [
C]NAD, with
the radiolabel present in the nicotinamide-proximal ribose, and with
[ adenylate-
P]NAD. ADP-ribose attached
to integrin
7 was then allowed to be degraded, as documented by
the loss of
P label (Fig. 8). Under these conditions,
[
C]ribose remained fully associated with
integrin
7, consistent with the degradation of ADP-ribose by a
cell surface 5`-nucleotide phosphodiesterase.
7 in skeletal
muscle myotubes was heterogenously modified in its extracellular
domain. GPI-anchored ADP-ribosyltransferase catalyzed the first step of
the modification, adding ADP-ribose to arginine residues in the
protein. Depending on the NAD concentration, different numbers of sites
in integrin
7 were modified. At micromolar NAD, a single site (or
perhaps several different sites not distinguishable by partial tryptic
digestion of the integrin and SDS-PAGE) located in the 39-kDa segment
between amino acids 575 and 886 was the preferred target for
ADP-ribosyltransferase. At higher NAD concentrations, the 63-kDa
N-terminal region of integrin
7 was apparently also modified. The
extent of ADP-ribosylation of integrin
7 did not seem to affect
its interaction with integrin
1 or the interactions of
7
1 dimer with cytoskeleton or the ECM protein laminin. An
effect of ADP-ribosylation on the structure of integrin
7 and its
other functions cannot, however, be excluded.
7, is subjected to rapid
degradation by a cell surface phosphodiesterase, leading to the
formation of phosphoribosylarginine in the protein, which probably
undergoes further dephosphorylation by extracellular phosphatases
(23, 25) . As a result of stepwise degradation of
ADP-ribose, integrin
7 is heterogenously modified, with each form
of modification having potentially a different effect on the structure
of the protein.
P label was almost completely
lost from ADP-ribosylated integrin
7, while
[
C]ribose remained fully associated with the
protein. Consistently, the sites in integrin
7, which were
previously ADP-ribosylated and then modified by phosphodiesterase
action, were effectively blocked for at least 1 h to subsequent
ADP-ribosylation, although the level of integrin
7 remained
constant at the cell surface. Thus, the turnover of extracellular
ADP-ribosylated protein may be different from that proposed for
intracellular ADP-ribosylated proteins
(3) . ADP-ribosylarginine
hydrolase, which is ubiquitously expressed in animal tissues
(20) , was proposed to exist with ADP-ribosyltransferases in an
ADP-ribosylation cycle
(3) . The location of the hydrolase
suggests that it may not participate in a modification cycle with the
GPI-linked transferase.
7 is a
physiologically important mechanism for regulation of this protein,
there should be a supply of NAD at the cell surface. This question has
not yet been addressed, but there are at least two other classes of
extracellular enzymes that use NAD: NAD glycohydrolases
(26) and ADP-ribose cyclases
(27, 28) . Given the
complex topography of the muscle fiber surface, the possibility of
local or transient availability of NAD does not seem unrealistic.
7 and the metabolism of ADP-ribose
linked to the integrin suggest that ADP-ribosyltransferase,
phosphodiesterase, and integrin
7 itself are in a close proximity
in the cell membrane. This is rather a surprising observation because
at least two of these proteins seem to be associated with different
membrane domains. Integrin
7, unless cross-linked with antibody
reactive with its extracellular domain, is loosely linked to the
cytoskeleton and is easily extracted with Triton X-100
(19) .
ADP-ribosyltransferase, as a GPI-anchored protein, may be located in
the glycolipid and cholesterol-rich membrane domains, characterized by
insolubility in Triton X-100
(29, 30, 31) . Cell
surface phosphodiesterase, reported to be a GPI-anchored protein in
some cells
(23) , was PI-PLC-resistant in C2C12 myotubes, and
its structure and localization are not clear. Even if the
ADP-ribosyltransferase and the modified integrin
7 molecules
reside in two different neighboring cells, their interaction seems very
specific. The findings reported here concerning the ADP-ribosylation of
integrin
7 should facilitate definition of the role of this
interaction in integrin function.
Table: Estimation of relative amounts of integrin 7 with different extent of ADP-ribosylation
P]NAD and 1 m
M ADP-ribose (ADPR). Following extraction with octyl glucoside and
purification on a laminin column, integrin
7 was analyzed by
SDS-PAGE. The relative amounts of the 102- and 105-kDa forms were
estimated after quantification of the intensity of silver staining of
the gels. The relative amounts of radioactivity associated with these
two forms were obtained after quantification with PhosphorImager. The
results for three different experiments are shown. Experiment 1
corresponds to that shown in Fig. 3.
C]NAD and Dr. Martha Vaughan
for critical review of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.