Characterization of the altered oligosaccharide composition of the insulin receptor on neural-derived cells.
Typical insulin receptors are present on neuroblastoma cell lines. High affinity binding for insulin was present in membrane preparations from NG108 (a hybrid mouse neuroblastoma-rat glioma) as well as in membranes from SK-N-MC and SK-N-SH, two human neuroblastoma cell lines. Specific [125I]insulin binding was 24.4% for NG108, 16.9% for SK-N-MC and 5.2% for SK-N-SH at membrane protein concentrations of 0.4 mg/ml. IC50 for [125I]insulin binding was 3.4 nM in NG108 membrane preparations and 0.9 nM for SK-N-SH and 1.8 nM in SK-N-MC membranes. Apparent mol. wt. for the alpha subunits (identified by specific immunoprecipitation using the anti-insulin receptor antiserum B10) on SDS PAGE was 134 kDa for NG108; 124 kDa for SK-N-MC and 120 kDa for SK-N-SH. Neuraminidase digestion increased the mobility of the alpha subunit from both NG108 and SK-N-MC receptors to 120 kDa, whereas that from SK-N-SH were unaffected. Endoglycosidase H and endoglycosidase F digestions increased the mobility of the alpha subunits of all 3 cell lines to varying degrees, suggesting the presence of N-linked glycosylation. Insulin induced autophosphorylation of the insulin receptor beta subunit in WGA-purified membranes from all 3 cell lines. In addition, phosphorylation of a protein with an apparent mol. wt. 105 kDa was stimulated by insulin in WGA purified membranes from NG108. Tyrosine-specific kinase activity was present in the membranes from each cell line and was stimulated by insulin in a dose-dependent manner from 10(-9) to 10(-6) M. Proinsulin was about 100 times less potent in stimulating phosphorylation of the artificial substrate poly (Glu, Tyr)4:1 when compared to insulin in accordance with its lower binding affinity to the insulin receptor. Hexose transport was stimulated by insulin in all 3 cell lines. These results indicate that neuroblastoma cells contain specific insulin receptors and that they may be useful as models for studying the role of insulin in nervous tissue.
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Section of Molecular and Cellular Physiology, NIDDK, Bethesda, MD 20892.
Shemer
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J
Pruss
R M
RM
Lowe
W L
WL
LeRoith
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D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003847:Deoxyglucose / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D009447:Neuroblastoma; D009844:Oligosaccharides / Q000302:isolation & purification*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D009844:Oligosaccharides; D003847:Deoxyglucose; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin