Biosynthesis and processing of the type II insulin-like growth factor receptor in H-35 hepatoma cells

R. G. MacDonald, M. P. Czech

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

The biosynthesis and post-translational processing of the insulin-like growth factor II (IGF-II) receptor has been studied in H-35 hepatoma cells using a specific polyclonal anti-receptor immunoglobulin preparation. Cells were pulse-labeled with [35S]methionine followed by incubation with excess unlabeled methionine (chase). Gel electrophoresis of the immunoadsorbed receptors shows that the receptor is first synthesized as a 245-kDa precursor which is transformed to the mature 250-kDa form with a half-time of about 2 h. The 245-kDa precursor could also be labeled biosynthetically with [3H]mannose, only one-half of which was ultimately found associated with the 250-kDa product. Neuraminidase converts the 250-kDa receptor species to a 245-kDa form. Whereas the 250-kDa receptor is insenitive to detectable cleavage by endoglycosidase H, digestion of the 245-kDa species with this enzyme produces a 232-kDa form. A similar 232-kDa receptor species accumulates in H-35 cells incubated with tunicamycin (2 μg/ml). This tunicamycin-induced aglyco-receptor is not further processed to the 250-kDa form. Monensin (50 nM) blocks receptor processing at the 245-kDa stage. Endoglycosidase H treatment of the monensin-induced 245-kDa species indicates that this is a mixture of partially processed precursors having equivalent M(r). No evidence was obtained for the presence of O-linked oligosaccharides on the IGF-II receptor. The IGF-II binding activity of the three different biosynthetic forms of the receptor was assessed by affinity cross-linking of 125I-IGF-II to the receptors using disuccinimidyl suberate. Both the mature 250-kDa receptor and the neuraminidase-digested 245-kDa form specifically bound 125I-IGF-II. However, the 232-kDa aglyco-receptor had no detectable IGF-II binding activity using this method. In summary, these studies show that the H-35 cell IGF-II receptor is synthesized first as a 245-kDa precursor having 4-6 high-mannose oligosaccharide side chains, processing of the receptor oligosaccharides by mannose removal and terminal sialylation converts the 245-kDa precursor to the 250-kDa mature product which has been previously identified as the functional receptor in the plasma membrane, the apparent molecular mass of the receptor in the absence of N-glycosylation is 232-kDa, and glycosylation of the IGF-II receptor is required for the acquisition of IGF-II binding activity.

Original languageEnglish (US)
Pages (from-to)11357-11365
Number of pages9
JournalJournal of Biological Chemistry
Volume260
Issue number20
StatePublished - Dec 1 1985

Fingerprint

IGF Type 2 Receptor
Insulin-Like Growth Factor II
Biosynthesis
Hepatocellular Carcinoma
Mannose
Oligosaccharides
Glycosylation
Tunicamycin
Processing
Monensin
Glycoside Hydrolases
Neuraminidase
Methionine
Molecular mass
Cell membranes
Electrophoresis
Immunoglobulins
Digestion
Gels
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Biosynthesis and processing of the type II insulin-like growth factor receptor in H-35 hepatoma cells. / MacDonald, R. G.; Czech, M. P.

In: Journal of Biological Chemistry, Vol. 260, No. 20, 01.12.1985, p. 11357-11365.

Research output: Contribution to journalArticle

@article{d12fe58074354ff6b921e01fc398e7e6,
title = "Biosynthesis and processing of the type II insulin-like growth factor receptor in H-35 hepatoma cells",
abstract = "The biosynthesis and post-translational processing of the insulin-like growth factor II (IGF-II) receptor has been studied in H-35 hepatoma cells using a specific polyclonal anti-receptor immunoglobulin preparation. Cells were pulse-labeled with [35S]methionine followed by incubation with excess unlabeled methionine (chase). Gel electrophoresis of the immunoadsorbed receptors shows that the receptor is first synthesized as a 245-kDa precursor which is transformed to the mature 250-kDa form with a half-time of about 2 h. The 245-kDa precursor could also be labeled biosynthetically with [3H]mannose, only one-half of which was ultimately found associated with the 250-kDa product. Neuraminidase converts the 250-kDa receptor species to a 245-kDa form. Whereas the 250-kDa receptor is insenitive to detectable cleavage by endoglycosidase H, digestion of the 245-kDa species with this enzyme produces a 232-kDa form. A similar 232-kDa receptor species accumulates in H-35 cells incubated with tunicamycin (2 μg/ml). This tunicamycin-induced aglyco-receptor is not further processed to the 250-kDa form. Monensin (50 nM) blocks receptor processing at the 245-kDa stage. Endoglycosidase H treatment of the monensin-induced 245-kDa species indicates that this is a mixture of partially processed precursors having equivalent M(r). No evidence was obtained for the presence of O-linked oligosaccharides on the IGF-II receptor. The IGF-II binding activity of the three different biosynthetic forms of the receptor was assessed by affinity cross-linking of 125I-IGF-II to the receptors using disuccinimidyl suberate. Both the mature 250-kDa receptor and the neuraminidase-digested 245-kDa form specifically bound 125I-IGF-II. However, the 232-kDa aglyco-receptor had no detectable IGF-II binding activity using this method. In summary, these studies show that the H-35 cell IGF-II receptor is synthesized first as a 245-kDa precursor having 4-6 high-mannose oligosaccharide side chains, processing of the receptor oligosaccharides by mannose removal and terminal sialylation converts the 245-kDa precursor to the 250-kDa mature product which has been previously identified as the functional receptor in the plasma membrane, the apparent molecular mass of the receptor in the absence of N-glycosylation is 232-kDa, and glycosylation of the IGF-II receptor is required for the acquisition of IGF-II binding activity.",
author = "MacDonald, {R. G.} and Czech, {M. P.}",
year = "1985",
month = "12",
day = "1",
language = "English (US)",
volume = "260",
pages = "11357--11365",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "20",

}

TY - JOUR

T1 - Biosynthesis and processing of the type II insulin-like growth factor receptor in H-35 hepatoma cells

AU - MacDonald, R. G.

AU - Czech, M. P.

PY - 1985/12/1

Y1 - 1985/12/1

N2 - The biosynthesis and post-translational processing of the insulin-like growth factor II (IGF-II) receptor has been studied in H-35 hepatoma cells using a specific polyclonal anti-receptor immunoglobulin preparation. Cells were pulse-labeled with [35S]methionine followed by incubation with excess unlabeled methionine (chase). Gel electrophoresis of the immunoadsorbed receptors shows that the receptor is first synthesized as a 245-kDa precursor which is transformed to the mature 250-kDa form with a half-time of about 2 h. The 245-kDa precursor could also be labeled biosynthetically with [3H]mannose, only one-half of which was ultimately found associated with the 250-kDa product. Neuraminidase converts the 250-kDa receptor species to a 245-kDa form. Whereas the 250-kDa receptor is insenitive to detectable cleavage by endoglycosidase H, digestion of the 245-kDa species with this enzyme produces a 232-kDa form. A similar 232-kDa receptor species accumulates in H-35 cells incubated with tunicamycin (2 μg/ml). This tunicamycin-induced aglyco-receptor is not further processed to the 250-kDa form. Monensin (50 nM) blocks receptor processing at the 245-kDa stage. Endoglycosidase H treatment of the monensin-induced 245-kDa species indicates that this is a mixture of partially processed precursors having equivalent M(r). No evidence was obtained for the presence of O-linked oligosaccharides on the IGF-II receptor. The IGF-II binding activity of the three different biosynthetic forms of the receptor was assessed by affinity cross-linking of 125I-IGF-II to the receptors using disuccinimidyl suberate. Both the mature 250-kDa receptor and the neuraminidase-digested 245-kDa form specifically bound 125I-IGF-II. However, the 232-kDa aglyco-receptor had no detectable IGF-II binding activity using this method. In summary, these studies show that the H-35 cell IGF-II receptor is synthesized first as a 245-kDa precursor having 4-6 high-mannose oligosaccharide side chains, processing of the receptor oligosaccharides by mannose removal and terminal sialylation converts the 245-kDa precursor to the 250-kDa mature product which has been previously identified as the functional receptor in the plasma membrane, the apparent molecular mass of the receptor in the absence of N-glycosylation is 232-kDa, and glycosylation of the IGF-II receptor is required for the acquisition of IGF-II binding activity.

AB - The biosynthesis and post-translational processing of the insulin-like growth factor II (IGF-II) receptor has been studied in H-35 hepatoma cells using a specific polyclonal anti-receptor immunoglobulin preparation. Cells were pulse-labeled with [35S]methionine followed by incubation with excess unlabeled methionine (chase). Gel electrophoresis of the immunoadsorbed receptors shows that the receptor is first synthesized as a 245-kDa precursor which is transformed to the mature 250-kDa form with a half-time of about 2 h. The 245-kDa precursor could also be labeled biosynthetically with [3H]mannose, only one-half of which was ultimately found associated with the 250-kDa product. Neuraminidase converts the 250-kDa receptor species to a 245-kDa form. Whereas the 250-kDa receptor is insenitive to detectable cleavage by endoglycosidase H, digestion of the 245-kDa species with this enzyme produces a 232-kDa form. A similar 232-kDa receptor species accumulates in H-35 cells incubated with tunicamycin (2 μg/ml). This tunicamycin-induced aglyco-receptor is not further processed to the 250-kDa form. Monensin (50 nM) blocks receptor processing at the 245-kDa stage. Endoglycosidase H treatment of the monensin-induced 245-kDa species indicates that this is a mixture of partially processed precursors having equivalent M(r). No evidence was obtained for the presence of O-linked oligosaccharides on the IGF-II receptor. The IGF-II binding activity of the three different biosynthetic forms of the receptor was assessed by affinity cross-linking of 125I-IGF-II to the receptors using disuccinimidyl suberate. Both the mature 250-kDa receptor and the neuraminidase-digested 245-kDa form specifically bound 125I-IGF-II. However, the 232-kDa aglyco-receptor had no detectable IGF-II binding activity using this method. In summary, these studies show that the H-35 cell IGF-II receptor is synthesized first as a 245-kDa precursor having 4-6 high-mannose oligosaccharide side chains, processing of the receptor oligosaccharides by mannose removal and terminal sialylation converts the 245-kDa precursor to the 250-kDa mature product which has been previously identified as the functional receptor in the plasma membrane, the apparent molecular mass of the receptor in the absence of N-glycosylation is 232-kDa, and glycosylation of the IGF-II receptor is required for the acquisition of IGF-II binding activity.

UR - http://www.scopus.com/inward/record.url?scp=0022214365&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0022214365&partnerID=8YFLogxK

M3 - Article

C2 - 2993308

AN - SCOPUS:0022214365

VL - 260

SP - 11357

EP - 11365

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 20

ER -