©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Processing of ADP-ribosylated Integrin 7 in Skeletal Muscle Myotubes (*)

Anna Zolkiewska (§) , Joel Moss

From the (1) Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1434

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Integrin 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.


INTRODUCTION

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)() -anchored protein (6, 7) located on the cell surface (7, 8) .

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 and subunits (9) .

Integrin 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.

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 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.


MATERIALS AND METHODS

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 10cells/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 CaCland 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 1

C2C12 cells were solubilized in 20 m M Tris-Cl (pH 7.5), 150 m M NaCl, 1 m M MnCl, 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 cmm 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 7

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 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.




RESULTS

Incubation of intact differentiated mouse skeletal muscle C2C12 myoblasts (myotubes) with [ adenylate-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).

To determine whether the 102- and 105-kDa ADP-ribosylated proteins represented different forms of integrin 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.

It is not clear why the mobilities of integrin 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.

The ratio of radioactivity associated with the 105- and 102-kDa bands after labeling of cells with 75 µ M [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%) ().

Following incubation of cells with 5 µ M [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 nmolminmg 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).

Radioactivity associated with integrin 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) .

The removal of [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.


DISCUSSION

In the presence of external NAD, integrin 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 71 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.

Extracellular ADP-ribose, free or linked to integrin 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.

(Phospho)ribosylarginine is much more stable than ADP-ribosylarginine in the extracellular compartment. 1 h after termination of the labeling, 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.

If ADP-ribosylation of integrin 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.

ADP-ribosylation of integrin 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

C2C12 myotubes (day 10 after plating) were incubated for 15 min in the presence of 75 µ M [ adenylate-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.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Bldg. 10, Rm. 5N307, 10 Center Rd., MSC 1434, National Institutes of Health, Bethesda, MD 20892-1434. Tel.: 301-496-5013; Fax: 301-402-1610.

The abbreviations used are: GPI, glycosylphosphatidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C; ECM, extracellular matrix; PAGE, polyacrylamide gel electrophoresis; DPBS, Dulbecco's phosphate-buffered saline; HPLC, high performance liquid chromatography; NP-TMP, p-nitrophenylthymidine-5`-monophosphate.


ACKNOWLEDGEMENTS

We thank Dr. Myron Jacobson for supplying [ ribose-U-C]NAD and Dr. Martha Vaughan for critical review of the manuscript.


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