Characterization of Interferon-α Binding Sites on Human Cell Lines

Catherine Vanden Broecke, Lawrence Pfeffer

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

The binding sites for human interferon-α (IFN-α) have been characterized on human lymphoblastoid, melanoma, rhabdomyosarcoma, and cervical carcinoma cells. Crosslinking of iodinated-recombinant DNA-derived IFN-α-Con1, an analog of the known IFN-α subtypes, to the cell surface with disuccinimidyl suberate yielded four IFN-receptor complexes of 118, 138, 159, and 260 kD on all cell lines that specifically bind IFN-α. Since IFN-α exists in solution as monomers, dimers, and trimers, and the three lower molecular weight IFN-α—receptor complexes differ by the molecular weight of IFN-α (20 kD), this suggests that the human IFN-α receptor of 100 kD binds more than one molecule of IFN-α. The higher molecular weight complex of 260 kD may result from dimerization of the receptor. None of these complexes was observed in a rhabdomyosarcoma subclone that does not specifically bind IFN-α. Pretreatment of cells with trypsin abolished the formation of these complexes. Pretreatment of cells with neuraminidase did not reduce IFN-α binding, but increased the electrophoretic mobility of all four IFN-α-receptor complexes. Other glycosidases (i.e., mannosidase, β-galactosidase, and endoglycosidase F) had no effects on IFN-α binding or mobility of complexes. Thus, although the IFN-α receptor is a glycoprotein, the glycosylated portion is apparently not part of the IFN-aα-binding domain. The formation of IFN-α—receptor complexes is independent of the duration of incubation with IFN (from 5 min to 1 h at 15°C). Subclones of Dandi lymphoblastoid cells that are sensitive (Cl-1) or resistant (Cl-2) to the antiproliferative effect of IFN-α have similar IFN-α binding capacity. The association of the IFN—receptor complexes with Triton-insoluble cytoskeletal matrix is markedly diminished in resistant Cl-2 cells relative to that in sensitive Cl-1 cells. However, IFN—receptor complexes, including those noncovalently associated with the cytoskeleton, are structurally similar in sensitive and resistant subclones. Thus, a post-binding defect may be responsible for the resistance of Cl-2 cells to the antiproliferative effect of IFN-α.

Original languageEnglish (US)
Pages (from-to)803-811
Number of pages9
JournalJournal of Interferon Research
Volume8
Issue number6
DOIs
StatePublished - Jan 1 1988
Externally publishedYes

Fingerprint

Interferons
Binding Sites
Cell Line
Interferon Receptors
Rhabdomyosarcoma
Molecular Weight
Mannosidases
Galactosidases
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
Recombinant DNA
Glycoside Hydrolases
Neuraminidase
Dimerization
Cytoskeleton
Trypsin
Melanoma
Glycoproteins
Carcinoma

All Science Journal Classification (ASJC) codes

  • Immunology
  • Virology

Cite this

Characterization of Interferon-α Binding Sites on Human Cell Lines. / Broecke, Catherine Vanden; Pfeffer, Lawrence.

In: Journal of Interferon Research, Vol. 8, No. 6, 01.01.1988, p. 803-811.

Research output: Contribution to journalArticle

@article{f8a0388a467c4d04a8c0eade6f424260,
title = "Characterization of Interferon-α Binding Sites on Human Cell Lines",
abstract = "The binding sites for human interferon-α (IFN-α) have been characterized on human lymphoblastoid, melanoma, rhabdomyosarcoma, and cervical carcinoma cells. Crosslinking of iodinated-recombinant DNA-derived IFN-α-Con1, an analog of the known IFN-α subtypes, to the cell surface with disuccinimidyl suberate yielded four IFN-receptor complexes of 118, 138, 159, and 260 kD on all cell lines that specifically bind IFN-α. Since IFN-α exists in solution as monomers, dimers, and trimers, and the three lower molecular weight IFN-α—receptor complexes differ by the molecular weight of IFN-α (20 kD), this suggests that the human IFN-α receptor of 100 kD binds more than one molecule of IFN-α. The higher molecular weight complex of 260 kD may result from dimerization of the receptor. None of these complexes was observed in a rhabdomyosarcoma subclone that does not specifically bind IFN-α. Pretreatment of cells with trypsin abolished the formation of these complexes. Pretreatment of cells with neuraminidase did not reduce IFN-α binding, but increased the electrophoretic mobility of all four IFN-α-receptor complexes. Other glycosidases (i.e., mannosidase, β-galactosidase, and endoglycosidase F) had no effects on IFN-α binding or mobility of complexes. Thus, although the IFN-α receptor is a glycoprotein, the glycosylated portion is apparently not part of the IFN-aα-binding domain. The formation of IFN-α—receptor complexes is independent of the duration of incubation with IFN (from 5 min to 1 h at 15°C). Subclones of Dandi lymphoblastoid cells that are sensitive (Cl-1) or resistant (Cl-2) to the antiproliferative effect of IFN-α have similar IFN-α binding capacity. The association of the IFN—receptor complexes with Triton-insoluble cytoskeletal matrix is markedly diminished in resistant Cl-2 cells relative to that in sensitive Cl-1 cells. However, IFN—receptor complexes, including those noncovalently associated with the cytoskeleton, are structurally similar in sensitive and resistant subclones. Thus, a post-binding defect may be responsible for the resistance of Cl-2 cells to the antiproliferative effect of IFN-α.",
author = "Broecke, {Catherine Vanden} and Lawrence Pfeffer",
year = "1988",
month = "1",
day = "1",
doi = "10.1089/jir.1988.8.803",
language = "English (US)",
volume = "8",
pages = "803--811",
journal = "Journal of Interferon and Cytokine Research",
issn = "1079-9907",
publisher = "Mary Ann Liebert Inc.",
number = "6",

}

TY - JOUR

T1 - Characterization of Interferon-α Binding Sites on Human Cell Lines

AU - Broecke, Catherine Vanden

AU - Pfeffer, Lawrence

PY - 1988/1/1

Y1 - 1988/1/1

N2 - The binding sites for human interferon-α (IFN-α) have been characterized on human lymphoblastoid, melanoma, rhabdomyosarcoma, and cervical carcinoma cells. Crosslinking of iodinated-recombinant DNA-derived IFN-α-Con1, an analog of the known IFN-α subtypes, to the cell surface with disuccinimidyl suberate yielded four IFN-receptor complexes of 118, 138, 159, and 260 kD on all cell lines that specifically bind IFN-α. Since IFN-α exists in solution as monomers, dimers, and trimers, and the three lower molecular weight IFN-α—receptor complexes differ by the molecular weight of IFN-α (20 kD), this suggests that the human IFN-α receptor of 100 kD binds more than one molecule of IFN-α. The higher molecular weight complex of 260 kD may result from dimerization of the receptor. None of these complexes was observed in a rhabdomyosarcoma subclone that does not specifically bind IFN-α. Pretreatment of cells with trypsin abolished the formation of these complexes. Pretreatment of cells with neuraminidase did not reduce IFN-α binding, but increased the electrophoretic mobility of all four IFN-α-receptor complexes. Other glycosidases (i.e., mannosidase, β-galactosidase, and endoglycosidase F) had no effects on IFN-α binding or mobility of complexes. Thus, although the IFN-α receptor is a glycoprotein, the glycosylated portion is apparently not part of the IFN-aα-binding domain. The formation of IFN-α—receptor complexes is independent of the duration of incubation with IFN (from 5 min to 1 h at 15°C). Subclones of Dandi lymphoblastoid cells that are sensitive (Cl-1) or resistant (Cl-2) to the antiproliferative effect of IFN-α have similar IFN-α binding capacity. The association of the IFN—receptor complexes with Triton-insoluble cytoskeletal matrix is markedly diminished in resistant Cl-2 cells relative to that in sensitive Cl-1 cells. However, IFN—receptor complexes, including those noncovalently associated with the cytoskeleton, are structurally similar in sensitive and resistant subclones. Thus, a post-binding defect may be responsible for the resistance of Cl-2 cells to the antiproliferative effect of IFN-α.

AB - The binding sites for human interferon-α (IFN-α) have been characterized on human lymphoblastoid, melanoma, rhabdomyosarcoma, and cervical carcinoma cells. Crosslinking of iodinated-recombinant DNA-derived IFN-α-Con1, an analog of the known IFN-α subtypes, to the cell surface with disuccinimidyl suberate yielded four IFN-receptor complexes of 118, 138, 159, and 260 kD on all cell lines that specifically bind IFN-α. Since IFN-α exists in solution as monomers, dimers, and trimers, and the three lower molecular weight IFN-α—receptor complexes differ by the molecular weight of IFN-α (20 kD), this suggests that the human IFN-α receptor of 100 kD binds more than one molecule of IFN-α. The higher molecular weight complex of 260 kD may result from dimerization of the receptor. None of these complexes was observed in a rhabdomyosarcoma subclone that does not specifically bind IFN-α. Pretreatment of cells with trypsin abolished the formation of these complexes. Pretreatment of cells with neuraminidase did not reduce IFN-α binding, but increased the electrophoretic mobility of all four IFN-α-receptor complexes. Other glycosidases (i.e., mannosidase, β-galactosidase, and endoglycosidase F) had no effects on IFN-α binding or mobility of complexes. Thus, although the IFN-α receptor is a glycoprotein, the glycosylated portion is apparently not part of the IFN-aα-binding domain. The formation of IFN-α—receptor complexes is independent of the duration of incubation with IFN (from 5 min to 1 h at 15°C). Subclones of Dandi lymphoblastoid cells that are sensitive (Cl-1) or resistant (Cl-2) to the antiproliferative effect of IFN-α have similar IFN-α binding capacity. The association of the IFN—receptor complexes with Triton-insoluble cytoskeletal matrix is markedly diminished in resistant Cl-2 cells relative to that in sensitive Cl-1 cells. However, IFN—receptor complexes, including those noncovalently associated with the cytoskeleton, are structurally similar in sensitive and resistant subclones. Thus, a post-binding defect may be responsible for the resistance of Cl-2 cells to the antiproliferative effect of IFN-α.

UR - http://www.scopus.com/inward/record.url?scp=0024214006&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024214006&partnerID=8YFLogxK

U2 - 10.1089/jir.1988.8.803

DO - 10.1089/jir.1988.8.803

M3 - Article

VL - 8

SP - 803

EP - 811

JO - Journal of Interferon and Cytokine Research

JF - Journal of Interferon and Cytokine Research

SN - 1079-9907

IS - 6

ER -