- Email: [email protected]
[48] Procedures for binding an antibody to receptor-bound interferon
326
INTERFERON RECEPTOR AND UPTAKE OF INTERFERON
[48]
[48] P r o c e d u r e s for B i n d i n g a n A n t i b o d y to Receptor-Bound Interferon
By HEINZ ARNHEITER and KATHRYN C. ZOON Antibodies recognizing receptor-bound IFNs are interesting for various reasons. They may be used to isolate IFN/receptor complexes, to analyze structure/function relationships of IFNs, to analyze the fate of IFNs bound to the surface of living cells, or to identify individual cells with particularly high or particularly low numbers of IFN receptors. Ideally, such antibodies should bind to receptor-bound IFN with high affinity, they should not displace IFN from its receptor, they should not interfere with IFN binding to receptors, and, consequently, not neutralize IFN activity. It appears that antibodies specific for one epitope (or a narrow range of epitopes) of IFNs may satisfy at least some of these criteria. Here, we briefly describe procedures for binding a mouse monoclonal IgG~ antibody, 111/21, to cell surface-bound IFN. This antibody was made against a synthetic, 56-residue carboxyl-terminal fragment of Hu-IFNa l.l The antibody binds to native recombinant DNA-derived IFN-a 1 and -a22 at the ultimate 10-16 carboxyl-terminal residues, and it binds both native Hu-IFN-a2 and the synthetic oligopeptide Hu-IFN-al (151-166) with an apparent Kd of 6.0 x 10-l° M. 3 It does not neutralize IFN unless used at a 106-fold molar excess over IFN, it does not inhibit the equilibrium binding of Hu-IFN-a2 to Madin-Darby bovine kidney (MDBK) cells unless added at a molar excess over IFN of at least 4000-fold, and it does not significantly displace Hu-IFN-a2 previously bound to cells under equilibrium conditions. 3 This suggests that the antibody recognizes IFN at a site which does not play a crucial role for receptor binding. Preparative Procedures and Methodology
Purification of Antibody 111/21 Ascitic fluids collected from female BALB/c mice into which 107 cells of hybridoma clone III/21 have been injected intraperitoneally are precipi H. Arnheiter, R. M. Thomas, T. Leist, M. Fountoulakis, and B. Gutte, Nature (London) 294, 278 (1981). : F o r some of the studies, H u - I F N - a A was used instead of Hu-IFN-a2. H u - I F N - a 2 differs from H u - I F N - a A at position 23 in the amino acid sequence; H u - I F N - a 2 has an arginine at this position, H u - I F N - a A has a lysine. 3 H. Arnheiter, M. Ohno, M. Smith, B. Gutte, and K. C. Zoon, Proc. Natl. Acad. Sci. U.S.A. 80, 2539 (1983).
METHODS IN ENZYMOLOGY,VOL. 119
Copyright © 1986by Academic Press, Inc. All rights of reproduction in any form reserved.
[48]
RECEPTOR-BOUND INTERFERON
327
itated at 4° with 50% ammonium sulfate. The precipitate is dissolved in and dialyzed against phosphate-buffered saline (PBS), pH 7.4. Approximately 30 mg of the dialyzed material is applied to an Affi-Gel 10 column (Bio-Rad) to which 20 mg of synthetic Hu-IFN-al (111-166) is coupled. Affinity columns to which other corresponding synthetic peptides or HuIFN-al (1-166) or Hu-IFN-~2 (1-165) are coupled may also be suitable. The column is washed with approximately 30 column volumes of PBS, pH 7.4, subsequently with 10 ml of Mcllvaine's citric acid/Na2HPO4 buffer at pH 6.0, and then with 10 ml of the same buffer at pH 4.5. Finally the antibody is eluted with 5 mi of the same buffer at pH 2.8. The eluate is dialyzed against PBS, pH 7.4, and stored frozen at -20 ° or kept at 4° in presence of 0.05% NAN3. With this procedure we could regularly recover in the pH 2.8 fraction approximately 25% of the protein applied to the column. The recovered antibody was of satisfactory purity (see below).
Radiolabeling of Antibody A portion of the purified antibody is dialyzed against 0.1 M sodium borate buffer, pH 8.5, and 100-200/zg (volume 50-100/~1) of the dialyzed antibody is added to a vial containing 1 mCi of ~zSI-labeled Bolton-Hunter reagent, for 15 min at room temperature. The reaction is stopped with 0.2 M glycine, pH 8.5, for 15 min. The radiolabeled antibody is separated from unreacted bolton-Hunter reagent by passage over a Sephadex G-25 column (Pharmacia) equilibrated with PBS, pH 7.4, and subsequent dialysis against PBS, pH 7.4. Purity of the lzSI-labeled antibody is checked by polyacrylamide gel electrophoresis as shown in Fig. 1. Its specific activity is determined by a standard enzyme-linked immunosorbent assay with synthetic Hu-IFN-al (111-166) or native Hu-IFN-a coated to plastic plates and a peroxidase-coupled anti-mouse IgG antibody. Unlabeled, purified monoclonal antibody III/21, whose concentration is determined by a Lowry assay, serves to establish the standard curve. We have obtained iodinated antibody preparations with specific activities between 0.7 and 1.5 × 1016 Bq/mol.
Purification and Radiolabeling of Hu-IFN-a2 Purified recombinant DNA-derived Hu-IFN-~2 (from Dr. Charles Weissmann, Zurich, Switzerland) or Hu-IFN-aA (Hoffmann-La Roche, Nutley, New Jersey) is labeled with ~25I-labeled Bolton-Hunter reagent as described? The specific activity of both labeled and unlabeled IFN against VSV in MDBK cells is 1.5-2.0 × 108 units/mg protein. 4 D. Zur Nedden and K. C. Zoon, this volume [38].
328
INTERFERON RECEPTOR AND UPTAKE OF INTERFERON
[48]
He
t.C
o
FIG. 1. Autoradiography of a 10% polyacrylamide gel in which purified, ~2~I-labeled antibody III/21 has been electrophoresed under reducing conditions. HC, Immunoglobulin heavy chain; LC, immunoglobulin light chain.
Cells
Madin-Darby bovine kidney cells (ATCC CCL 22) are cultured in Dulbecco's modified EMEM supplemented with 10% fetal calf serum and 50 tzg/ml of gentamicin sulfate (complete medium). Experiments are done with cells grown to confluent monolayers in 50-cm 2 plastic dishes (Falcon) at 37 ° (5-6 × 106 cells/dish).
[48]
329
RECEPTOR-BOUND INTERFERON
,~"
4.0-
N
~
-6.0
o
-4.0
o
3.0-
o 2.0-
o o
-2.0 1.0-
0
I
I
I
100
200
300
IFN
1250
a d d e d (pM)
FIG. 2. Binding of 1251-labeled antibody Ili/21 to Hu-IFN-a2 bound to the surface of monolayer MDBK cells. The cells were treated for 2.5 hr at 4° with unlabeled IFN at the concentrations indicated. The antibody (0.14 nM) was added for another 2 hr (©). Antibody neutralized with Hu-IFN-al (151-166), 1.3/xM (0). Antibody bound to plates with no cells (11). Each point represents the mean of the results from six independent culture dishes (see Ref. 3).
Binding of the Radiolabeled Monoclonal Antibody to Cell Surface-Bound IFN To test the binding of the radiolabeled antibody to cell surface-bound IFN, we add unlabeled IFN dissolved in 10 ml of ice cold complete medium to MDBK cells for 2.5 hr at 4°. The cells are then washed five times with ice cold PBS, pH 7.4, and exposed to radiolabeled antibody (0.14 nM in 10 ml of ice cold PBS, pH 7.4) for 2 hr at 4°. Then, the cells are washed five times with ice cold PBS, pH 7.4, solubilized with 0.5% sodium dodecyl sulfate in PBS, pH 7.4. The samples are then counted in a Beckman 8500 gamma counter. If antibody 111/21 recognized surface-bound Hu-IFN-a2, binding of antibody should reflect the dose dependent binding of IFN to MDBK cells. 5 This appears to be the case. For the representative experiment shown in Fig. 2, unlabeled Hu-IFN-o~2 was added to MDBK cells at concentrations between 0 and 1.25 nM and, in a parallel experiment, the amount of IFN bound to the cell surface was controlled by establishing a 5 K. C. Zoon, D. Zur Nedden, and H. Arnheiter, this volume [45].
330
INTERFERON RECEPTOR AND UPTAKE OF INTERFERON
[48]
binding curve with z25I-labeled Hu-IFN-a2. Each point in Fig. 2 represents the mean value obtained from six individual dishes. Antibody binding to cells reaches half saturation when half saturating IFN concentrations have previously been added to the cells, and reaches saturation when saturating IFN doses have been added (for binding curves of 1251labeled Hu-IF~q-a2 to MDBK cells, see Ref. 5). Over the concentration ranges tested, nonspecific binding of nSI-labeled IFN was less than 10% of the total of the radiolabeled IFN bound to the cell-surface. The molar amounts of cell-bound IFN and IFN-bound antibody are approximately the same. For instance, as shown in Fig. 2, the amount of radiolabeled antibody bound to 7 × 10 6 MDBK cells to which IFN had been added at a concentration of 250 pM is 5.5 fmol. Under the same conditions, the cells bound 5.8 fmol of nSI-labeled IFN specifically. To test the specificity of antibody binding the synthetic oligopeptide Hu-IFN-al (151-166) was used in direct competition assays (closed circle in Fig. 2), and the radiolabeled antibody was added to dishes to which IFN has been added in the absence of cells (close square in Fig. 2). In both controls, antibody binding is as low as its binding to cells not treated with IFN. Antibody III/21 Allows Determination of Changes in the Amount of Cell Surface-Bound IFN Occurring at 37 ° Radiolabeled Hu-IFN-a2 bound to the surface of MDBK cells at 4° is rapidly internalized by these cells when they are shifted to 370. 6 The corresponding decrease of surface-bound IFN should be detectable with the radiolabeled antibody III/21. To determine the amount of IFN remaining associated with the cell surface after incubation at 37°, the cells are washed five times with ice cold PBS and exposed to the radiolabeled antibody in the cold as described above. A representative experiment is shown in Fig. 3. At time 0, before the cells have been shifted to the warmer temperature, the total of the bound antibody was 3.3 fmol. With increasing time at 37 °, the amount of radiolabeled antibody bound to IFN decreased. To determine the amount of nSI-labeled IFN bound to the cell surface before and after the temperature shift, a parallel set of cells is exposed to radiolabeled IFN (125 pM in complete medium), and incubated in the cold for 2.5 hr and shifted to 37° as above. Then, the cells are quickly cooled to 4° and washed five times with ice cold PBS, pH 7.4, and exposed to 0.2 M acetic acid, 0.5 M NaC1, pH 2.5, for 10 min at 4°. This procedure removes surface-bound IFN and leaves internalized IFN asso6 K. C. Zoon, H. Arnheiter, and D. J. P. FitzGerald, this volume I49].
[48]
RECEPTOR-BOUND INTERFERON
331
4
m
o
E
3--
c o 1D 0
2--
r~
.m
o
0
I 10
I 20
I 30
t i m e at 3 7 ° C , min FIG. 3. Decrease of Hu-IFN-a2 on the surface of MDBK cells following incubation at 37°. Unlabeled IFN (125 pM) was added to cells at 4° for 2.5 hr. The cells were washed, incubated at 37° for the time periods indicated, washed again, exposed to 125I-labeled antibody (0.14 nM) for 2 hr at 4°, and cell-associated antibody was assayed (O). Antibody bound to cells not exposed to IFN (0).
ciated with the cells. 6'7 For the experiment shown above, the amount of radiolabeled IFN bound to the cell surface was 3.5 fmol before the shift, and it decreased to approximately 1.5 fmol 30 min after the shift. Thus, following incubation at 37 °, the decrease in acid-releasable material and the decrease in antibody binding are comparable.
Comments
Although the above procedure for binding an antibody to the carboxyl terminus of receptor-bound IFN appears straightforward, there is no guarantee that other monoclonal or polyclonal antibodies recognizing the 7 K. C. Zoon, H. Arnheiter, D. Zur Nedden, D. J. P. FitzGerald, and M. C. Willingham, Virology 130, 195 (1983).
332
INTERFERON
RECEPTOR AND UPTAKE OF INTERFERON
[49]
ultimate carboxyl terminus of IFN are able to recognize receptor-bound IFN. It is conceivable that IFN undergoes several conformational changes after binding to its specific receptor, and that IFN with such conformational changes is recognized only by particular antibodies. Antibody 111/21 may be unique in that it binds stoichiometrically to IFN bound to the surface of MDBK cells. It appears that it binds to both that portion of the IFN which is bound specifically to receptors as well as that portion which is bound nonspecifically to the cells.
[49] P r o c e d u r e s for M e a s u r i n g R e c e p t o r - M e d i a t e d B i n d i n g and Internalization of Human Interferon B y KATHRYN C. ZOON, HEINZ ARNHEITER, and DAVID FITZGERALD
The first step in the action of many polypeptide ligands is the specific binding of the ligand to its receptor on the plasma membrane. The receptor-bound ligand is then either taken up by the cell or processed and/or released at the cell surface. Monoclonal antibodies against the ligands or receptors, radioactive or fluorescent derivatives of ligands, and electrondense ligand conjugates have been successfully used to study the fate of the receptor-bound ligands and their receptors. Many polypeptide hormones, toxins, and other molecules such as growth factors and low-density lipoprotein are internalized by concentrative adsorptive endocytosis (receptor-mediated endocytosis) following the binding of ligands to their specific cell surface receptors. This pathway consists of clustering of the receptor-ligand complexes and transfer of the complexes to intracellular compartments (receptosomes, the Golgi system, and lysosomes; for reviews, see Refs. 1-3). Human interferons (IFNs) like many other polypeptide hormones interact with specific cell surface receptors and the complexes appear to be internalized. 4-8 In addition, at least a portion of human leukocyte interi j. L. Goldstein, R. G. W. Anderson, and M. S. Brown, Nature (London) 279, 679 (1979). 2 R. M. Steinman, I. S. Mellman, W. A. Muller, and Z. A. Cohn, J. CellBiol. 96, 1 (1983). 3 I. H. Pastan and M. C. Willingham, Science 214, 504 (1981). 4 K. C. Zoon, H. Arnheiter, D. Zur Nedden, D. J. P. FitzGerald, and M. C. Willingham, Virology 130, 195 (1983). 5 p. Anderson, Y. K. Yip, and J. Vil6ek, J. Biol. Chem. 258, 6497 (1983). 6 A. A. Branca, C. R. Faltynek, S. B. D'Alessandro, and C. Baglioni, J. Biol. Chem. 257, 13291 (1982). S. Yonehara, A. Ishii, and M. Yonehara-Takahashi, J. Gen. Virol. 64, 2409 (1983). s F. H. Sarkar and S. L. Gupta, Eur. J. Biochem. 140, 461 (1984).
METHODS IN ENZYMOLOGY, VOL. 119
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
INTERFERON RECEPTOR AND UPTAKE OF INTERFERON
[48]
[48] P r o c e d u r e s for B i n d i n g a n A n t i b o d y to Receptor-Bound Interferon
By HEINZ ARNHEITER and KATHRYN C. ZOON Antibodies recognizing receptor-bound IFNs are interesting for various reasons. They may be used to isolate IFN/receptor complexes, to analyze structure/function relationships of IFNs, to analyze the fate of IFNs bound to the surface of living cells, or to identify individual cells with particularly high or particularly low numbers of IFN receptors. Ideally, such antibodies should bind to receptor-bound IFN with high affinity, they should not displace IFN from its receptor, they should not interfere with IFN binding to receptors, and, consequently, not neutralize IFN activity. It appears that antibodies specific for one epitope (or a narrow range of epitopes) of IFNs may satisfy at least some of these criteria. Here, we briefly describe procedures for binding a mouse monoclonal IgG~ antibody, 111/21, to cell surface-bound IFN. This antibody was made against a synthetic, 56-residue carboxyl-terminal fragment of Hu-IFNa l.l The antibody binds to native recombinant DNA-derived IFN-a 1 and -a22 at the ultimate 10-16 carboxyl-terminal residues, and it binds both native Hu-IFN-a2 and the synthetic oligopeptide Hu-IFN-al (151-166) with an apparent Kd of 6.0 x 10-l° M. 3 It does not neutralize IFN unless used at a 106-fold molar excess over IFN, it does not inhibit the equilibrium binding of Hu-IFN-a2 to Madin-Darby bovine kidney (MDBK) cells unless added at a molar excess over IFN of at least 4000-fold, and it does not significantly displace Hu-IFN-a2 previously bound to cells under equilibrium conditions. 3 This suggests that the antibody recognizes IFN at a site which does not play a crucial role for receptor binding. Preparative Procedures and Methodology
Purification of Antibody 111/21 Ascitic fluids collected from female BALB/c mice into which 107 cells of hybridoma clone III/21 have been injected intraperitoneally are precipi H. Arnheiter, R. M. Thomas, T. Leist, M. Fountoulakis, and B. Gutte, Nature (London) 294, 278 (1981). : F o r some of the studies, H u - I F N - a A was used instead of Hu-IFN-a2. H u - I F N - a 2 differs from H u - I F N - a A at position 23 in the amino acid sequence; H u - I F N - a 2 has an arginine at this position, H u - I F N - a A has a lysine. 3 H. Arnheiter, M. Ohno, M. Smith, B. Gutte, and K. C. Zoon, Proc. Natl. Acad. Sci. U.S.A. 80, 2539 (1983).
METHODS IN ENZYMOLOGY,VOL. 119
Copyright © 1986by Academic Press, Inc. All rights of reproduction in any form reserved.
[48]
RECEPTOR-BOUND INTERFERON
327
itated at 4° with 50% ammonium sulfate. The precipitate is dissolved in and dialyzed against phosphate-buffered saline (PBS), pH 7.4. Approximately 30 mg of the dialyzed material is applied to an Affi-Gel 10 column (Bio-Rad) to which 20 mg of synthetic Hu-IFN-al (111-166) is coupled. Affinity columns to which other corresponding synthetic peptides or HuIFN-al (1-166) or Hu-IFN-~2 (1-165) are coupled may also be suitable. The column is washed with approximately 30 column volumes of PBS, pH 7.4, subsequently with 10 ml of Mcllvaine's citric acid/Na2HPO4 buffer at pH 6.0, and then with 10 ml of the same buffer at pH 4.5. Finally the antibody is eluted with 5 mi of the same buffer at pH 2.8. The eluate is dialyzed against PBS, pH 7.4, and stored frozen at -20 ° or kept at 4° in presence of 0.05% NAN3. With this procedure we could regularly recover in the pH 2.8 fraction approximately 25% of the protein applied to the column. The recovered antibody was of satisfactory purity (see below).
Radiolabeling of Antibody A portion of the purified antibody is dialyzed against 0.1 M sodium borate buffer, pH 8.5, and 100-200/zg (volume 50-100/~1) of the dialyzed antibody is added to a vial containing 1 mCi of ~zSI-labeled Bolton-Hunter reagent, for 15 min at room temperature. The reaction is stopped with 0.2 M glycine, pH 8.5, for 15 min. The radiolabeled antibody is separated from unreacted bolton-Hunter reagent by passage over a Sephadex G-25 column (Pharmacia) equilibrated with PBS, pH 7.4, and subsequent dialysis against PBS, pH 7.4. Purity of the lzSI-labeled antibody is checked by polyacrylamide gel electrophoresis as shown in Fig. 1. Its specific activity is determined by a standard enzyme-linked immunosorbent assay with synthetic Hu-IFN-al (111-166) or native Hu-IFN-a coated to plastic plates and a peroxidase-coupled anti-mouse IgG antibody. Unlabeled, purified monoclonal antibody III/21, whose concentration is determined by a Lowry assay, serves to establish the standard curve. We have obtained iodinated antibody preparations with specific activities between 0.7 and 1.5 × 1016 Bq/mol.
Purification and Radiolabeling of Hu-IFN-a2 Purified recombinant DNA-derived Hu-IFN-~2 (from Dr. Charles Weissmann, Zurich, Switzerland) or Hu-IFN-aA (Hoffmann-La Roche, Nutley, New Jersey) is labeled with ~25I-labeled Bolton-Hunter reagent as described? The specific activity of both labeled and unlabeled IFN against VSV in MDBK cells is 1.5-2.0 × 108 units/mg protein. 4 D. Zur Nedden and K. C. Zoon, this volume [38].
328
INTERFERON RECEPTOR AND UPTAKE OF INTERFERON
[48]
He
t.C
o
FIG. 1. Autoradiography of a 10% polyacrylamide gel in which purified, ~2~I-labeled antibody III/21 has been electrophoresed under reducing conditions. HC, Immunoglobulin heavy chain; LC, immunoglobulin light chain.
Cells
Madin-Darby bovine kidney cells (ATCC CCL 22) are cultured in Dulbecco's modified EMEM supplemented with 10% fetal calf serum and 50 tzg/ml of gentamicin sulfate (complete medium). Experiments are done with cells grown to confluent monolayers in 50-cm 2 plastic dishes (Falcon) at 37 ° (5-6 × 106 cells/dish).
[48]
329
RECEPTOR-BOUND INTERFERON
,~"
4.0-
N
~
-6.0
o
-4.0
o
3.0-
o 2.0-
o o
-2.0 1.0-
0
I
I
I
100
200
300
IFN
1250
a d d e d (pM)
FIG. 2. Binding of 1251-labeled antibody Ili/21 to Hu-IFN-a2 bound to the surface of monolayer MDBK cells. The cells were treated for 2.5 hr at 4° with unlabeled IFN at the concentrations indicated. The antibody (0.14 nM) was added for another 2 hr (©). Antibody neutralized with Hu-IFN-al (151-166), 1.3/xM (0). Antibody bound to plates with no cells (11). Each point represents the mean of the results from six independent culture dishes (see Ref. 3).
Binding of the Radiolabeled Monoclonal Antibody to Cell Surface-Bound IFN To test the binding of the radiolabeled antibody to cell surface-bound IFN, we add unlabeled IFN dissolved in 10 ml of ice cold complete medium to MDBK cells for 2.5 hr at 4°. The cells are then washed five times with ice cold PBS, pH 7.4, and exposed to radiolabeled antibody (0.14 nM in 10 ml of ice cold PBS, pH 7.4) for 2 hr at 4°. Then, the cells are washed five times with ice cold PBS, pH 7.4, solubilized with 0.5% sodium dodecyl sulfate in PBS, pH 7.4. The samples are then counted in a Beckman 8500 gamma counter. If antibody 111/21 recognized surface-bound Hu-IFN-a2, binding of antibody should reflect the dose dependent binding of IFN to MDBK cells. 5 This appears to be the case. For the representative experiment shown in Fig. 2, unlabeled Hu-IFN-o~2 was added to MDBK cells at concentrations between 0 and 1.25 nM and, in a parallel experiment, the amount of IFN bound to the cell surface was controlled by establishing a 5 K. C. Zoon, D. Zur Nedden, and H. Arnheiter, this volume [45].
330
INTERFERON RECEPTOR AND UPTAKE OF INTERFERON
[48]
binding curve with z25I-labeled Hu-IFN-a2. Each point in Fig. 2 represents the mean value obtained from six individual dishes. Antibody binding to cells reaches half saturation when half saturating IFN concentrations have previously been added to the cells, and reaches saturation when saturating IFN doses have been added (for binding curves of 1251labeled Hu-IF~q-a2 to MDBK cells, see Ref. 5). Over the concentration ranges tested, nonspecific binding of nSI-labeled IFN was less than 10% of the total of the radiolabeled IFN bound to the cell-surface. The molar amounts of cell-bound IFN and IFN-bound antibody are approximately the same. For instance, as shown in Fig. 2, the amount of radiolabeled antibody bound to 7 × 10 6 MDBK cells to which IFN had been added at a concentration of 250 pM is 5.5 fmol. Under the same conditions, the cells bound 5.8 fmol of nSI-labeled IFN specifically. To test the specificity of antibody binding the synthetic oligopeptide Hu-IFN-al (151-166) was used in direct competition assays (closed circle in Fig. 2), and the radiolabeled antibody was added to dishes to which IFN has been added in the absence of cells (close square in Fig. 2). In both controls, antibody binding is as low as its binding to cells not treated with IFN. Antibody III/21 Allows Determination of Changes in the Amount of Cell Surface-Bound IFN Occurring at 37 ° Radiolabeled Hu-IFN-a2 bound to the surface of MDBK cells at 4° is rapidly internalized by these cells when they are shifted to 370. 6 The corresponding decrease of surface-bound IFN should be detectable with the radiolabeled antibody III/21. To determine the amount of IFN remaining associated with the cell surface after incubation at 37°, the cells are washed five times with ice cold PBS and exposed to the radiolabeled antibody in the cold as described above. A representative experiment is shown in Fig. 3. At time 0, before the cells have been shifted to the warmer temperature, the total of the bound antibody was 3.3 fmol. With increasing time at 37 °, the amount of radiolabeled antibody bound to IFN decreased. To determine the amount of nSI-labeled IFN bound to the cell surface before and after the temperature shift, a parallel set of cells is exposed to radiolabeled IFN (125 pM in complete medium), and incubated in the cold for 2.5 hr and shifted to 37° as above. Then, the cells are quickly cooled to 4° and washed five times with ice cold PBS, pH 7.4, and exposed to 0.2 M acetic acid, 0.5 M NaC1, pH 2.5, for 10 min at 4°. This procedure removes surface-bound IFN and leaves internalized IFN asso6 K. C. Zoon, H. Arnheiter, and D. J. P. FitzGerald, this volume I49].
[48]
RECEPTOR-BOUND INTERFERON
331
4
m
o
E
3--
c o 1D 0
2--
r~
.m
o
0
I 10
I 20
I 30
t i m e at 3 7 ° C , min FIG. 3. Decrease of Hu-IFN-a2 on the surface of MDBK cells following incubation at 37°. Unlabeled IFN (125 pM) was added to cells at 4° for 2.5 hr. The cells were washed, incubated at 37° for the time periods indicated, washed again, exposed to 125I-labeled antibody (0.14 nM) for 2 hr at 4°, and cell-associated antibody was assayed (O). Antibody bound to cells not exposed to IFN (0).
ciated with the cells. 6'7 For the experiment shown above, the amount of radiolabeled IFN bound to the cell surface was 3.5 fmol before the shift, and it decreased to approximately 1.5 fmol 30 min after the shift. Thus, following incubation at 37 °, the decrease in acid-releasable material and the decrease in antibody binding are comparable.
Comments
Although the above procedure for binding an antibody to the carboxyl terminus of receptor-bound IFN appears straightforward, there is no guarantee that other monoclonal or polyclonal antibodies recognizing the 7 K. C. Zoon, H. Arnheiter, D. Zur Nedden, D. J. P. FitzGerald, and M. C. Willingham, Virology 130, 195 (1983).
332
INTERFERON
RECEPTOR AND UPTAKE OF INTERFERON
[49]
ultimate carboxyl terminus of IFN are able to recognize receptor-bound IFN. It is conceivable that IFN undergoes several conformational changes after binding to its specific receptor, and that IFN with such conformational changes is recognized only by particular antibodies. Antibody 111/21 may be unique in that it binds stoichiometrically to IFN bound to the surface of MDBK cells. It appears that it binds to both that portion of the IFN which is bound specifically to receptors as well as that portion which is bound nonspecifically to the cells.
[49] P r o c e d u r e s for M e a s u r i n g R e c e p t o r - M e d i a t e d B i n d i n g and Internalization of Human Interferon B y KATHRYN C. ZOON, HEINZ ARNHEITER, and DAVID FITZGERALD
The first step in the action of many polypeptide ligands is the specific binding of the ligand to its receptor on the plasma membrane. The receptor-bound ligand is then either taken up by the cell or processed and/or released at the cell surface. Monoclonal antibodies against the ligands or receptors, radioactive or fluorescent derivatives of ligands, and electrondense ligand conjugates have been successfully used to study the fate of the receptor-bound ligands and their receptors. Many polypeptide hormones, toxins, and other molecules such as growth factors and low-density lipoprotein are internalized by concentrative adsorptive endocytosis (receptor-mediated endocytosis) following the binding of ligands to their specific cell surface receptors. This pathway consists of clustering of the receptor-ligand complexes and transfer of the complexes to intracellular compartments (receptosomes, the Golgi system, and lysosomes; for reviews, see Refs. 1-3). Human interferons (IFNs) like many other polypeptide hormones interact with specific cell surface receptors and the complexes appear to be internalized. 4-8 In addition, at least a portion of human leukocyte interi j. L. Goldstein, R. G. W. Anderson, and M. S. Brown, Nature (London) 279, 679 (1979). 2 R. M. Steinman, I. S. Mellman, W. A. Muller, and Z. A. Cohn, J. CellBiol. 96, 1 (1983). 3 I. H. Pastan and M. C. Willingham, Science 214, 504 (1981). 4 K. C. Zoon, H. Arnheiter, D. Zur Nedden, D. J. P. FitzGerald, and M. C. Willingham, Virology 130, 195 (1983). 5 p. Anderson, Y. K. Yip, and J. Vil6ek, J. Biol. Chem. 258, 6497 (1983). 6 A. A. Branca, C. R. Faltynek, S. B. D'Alessandro, and C. Baglioni, J. Biol. Chem. 257, 13291 (1982). S. Yonehara, A. Ishii, and M. Yonehara-Takahashi, J. Gen. Virol. 64, 2409 (1983). s F. H. Sarkar and S. L. Gupta, Eur. J. Biochem. 140, 461 (1984).
METHODS IN ENZYMOLOGY, VOL. 119
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.