scholarly journals Increased levels of insulin and insulin-like growth factor-1 hybrid receptors and decreased glycosylation of the insulin receptor alpha- and beta-subunits in scrapie-infected neuroblastoma N2a cells

2004 ◽  
Vol 380 (2) ◽  
pp. 571-579 ◽  
Author(s):  
Daniel NIELSEN ◽  
Hanna GYLLBERG ◽  
Pernilla ÖSTLUND ◽  
Tomas BERGMAN ◽  
Katarina BEDECS
Keyword(s):  
Growth Factor ◽  
Insulin Receptor ◽  
Binding Sites ◽  
Cellular Localization ◽  
Fold Increase ◽  
Insulin Binding ◽  
Proteolytic Processing ◽  
Insulin Like Growth Factor ◽  
N2a Cells ◽  
Α And Β Subunits

We have previously shown that ScN2a cells (scrapie-infected neuroblastoma N2a cells) express 2-fold- and 4-fold-increased levels of IR (insulin receptor) and IGF-1R (insulin-like growth factor-1 receptor) respectively. In addition, the IR α- and β-subunits are aberrantly processed, with apparent molecular masses of 128 and 85 kDa respectively, as compared with 136 and 95 kDa in uninfected N2a cells. Despite the 2-fold increase in IR protein, the number of 125I-insulin-binding sites was slightly decreased in ScN2a cells [Östlund, Lindegren, Pettersson and Bedecs (2001) Brain Res. 97, 161–170]. In order to determine the cellular localization of IR in ScN2a cells, surface biotinylation was performed, showing a correct IR trafficking and localization to the cell surface. The present study shows for the first time that neuroblastoma N2a cells express significant levels of IR–IGF-1R hybrid receptors, and in ScN2a cells the number of hybrid receptors was 2-fold higher than that found in N2a cells, potentially explaining the apparent loss of insulin-binding sites due to a lower affinity for insulin compared with the homotypic IR. Furthermore, the decreased molecular mass of IR subunits in ScN2a cells is not caused by altered phosphorylation or proteolytic processing, but rather by altered glycosylation. Enzymic deglycosylation of immunoprecipitated IR from N2a and ScN2a cells with endoglycosidase H, peptide N-glycosidase F and neuraminidase all resulted in subunits with increased electrophoretic mobility; however, the 8–10 kDa shift remained. Combined enzymic or chemical deglycosylation using anhydrous trifluoromethane sulphonic acid treatment ultimately showed that the IR α- and β-subunits from ScN2a cells are aberrantly glycosylated. The increased formation of IR–IGF-1R hybrids in ScN2a cells may be part of a neuroprotective response to prion infection. The degree and functional significance of aberrantly glycosylated proteins in ScN2a cells remain to be determined.

scholarly journals Insulin-like growth factor binding to the atypical insulin receptors of a human lymphoid-derived cell line (IM-9)

1990 ◽  
Vol 266 (3) ◽  
pp. 737-742 ◽  
Author(s):  
H A Jonas ◽  
A J Cox
Keyword(s):  
Growth Factor ◽  
Affinity Chromatography ◽  
Insulin Receptor ◽  
Binding Sites ◽  
Insulin Binding ◽  
Type I ◽  
Insulin Like Growth Factor ◽  
Igf Receptors ◽  
Type I Igf Receptor ◽  
Igf I

The cells of the IM-9 human lymphocyte-derived line contain a sub-population of insulin-binding sites whose immunological and hormone-binding characteristics closely resemble those of the atypical insulin-binding sites of human placenta. These binding sites, which have moderately high affinity for multiplication-stimulating activity [MSA, the rat homologue of insulin-like growth factor (IGF) II] and IGF-I, are identified on IM-9 cells by 125I-MSA binding. They account for approximately 30% of the total insulin-receptor population, and do not react with a monoclonal antibody to the type I IGF receptor (alpha IR-3). The relative concentrations of unlabelled insulin, MSA and IGF-I required to displace 50% of 125I-MSA from these binding sites (1:4.7:29 respectively) are maintained for cells, particulate membranes, Triton-solubilized membranes precipitated either by poly(ethylene glycol) or a polyclonal antibody (B-10) to the insulin receptor, and receptors purified by insulin affinity chromatography. Because the atypical insulin/MSA-binding sites outnumber the type I IGF receptors in IM-9 cells by approximately 10-fold, they also compete with the latter receptors for 125I-IGF-I binding. Thus 125I-IGF-I binding to IM-9 cells is inhibited by moderately low concentrations of insulin (relative potency ratios for insulin compared with IGF-I are approx. 1/14 to 1/4) and is partially displaced (65-80%) by alpha IR-3. When type I IGF receptors are blocked by alpha IR-3 or removed by B-10 immunoprecipitation or insulin affinity chromatography, the hormone-displacement patterns for 125I-IGF-I binding resemble those of the atypical insulin/MSA-binding sites.

scholarly journals Insulin receptor is phosphorylated in response to treatment of HepG2 cells with insulin-like growth factor I

1990 ◽  
Vol 270 (1) ◽  
pp. 27-32 ◽  
Author(s):  
V Duronio
Keyword(s):  
Growth Factor ◽  
Insulin Receptor ◽  
Hepg2 Cells ◽  
Insulin Binding ◽  
Response To Treatment ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Igf I ◽  
Growth Factor I ◽  
In Cells

1. Binding of insulin and insulin-like growth factor I (IGF-I) to HepG2 cells was analysed with regard to competition by both insulin and IGF-I. At concentrations of insulin that caused maximal phosphorylation of the insulin receptor, virtually no displacement of IGF-I binding was observed. Similarly, at concentrations of IGF-I that caused maximal phosphorylation of the IGF-I receptor, no displacement of insulin binding was observed. 2. When the phosphorylation of both receptors was examined individually by using specific monoclonal antibodies to immunoprecipitate the receptors, phosphorylation of the insulin receptor was found to increase on both serine and tyrosine residues in cells treated with 100 ng of IGF-I/ml. In contrast, no increased phosphorylation of IGF-I receptor was observed in cells treated with 100 ng of insulin/ml. 3. The increase in phosphorylation of insulin receptor in response to IGF-I correlated with the dose-response of IGF-I-stimulated phosphorylation of the IGF-I receptor. 4. The IGF-I-stimulated phosphorylation of the insulin receptor could be blocked by preincubation with a monoclonal antibody that blocks IGF-I binding to the IGF-I receptor.

scholarly journals Expression and function of insulin/insulin-like growth factor I hybrid receptors during differentiation of 3T3-L1 preadipocytes

1998 ◽  
Vol 333 (3) ◽  
pp. 825-831 ◽  
Author(s):  
Dalit MODAN-MOSES ◽  
Michel JANICOT ◽  
John C. McLENITHAN ◽  
M. Daniel LANE ◽  
Samuel J. CASELLA
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Fold Increase ◽  
Insulin Binding ◽  
Cell Surface Expression ◽  
Insulin Like Growth Factor ◽  
Hybrid Form ◽  
Igf Receptors ◽  
Igf I ◽  
Igf Receptor

During the assembly of cell surface receptors, insulin proreceptors are sometimes joined to insulin-like growth factor (IGF) receptor precursors to form covalently linked hybrid receptors. To address the biological consequences of hybrid receptor formation, we studied 3T3-L1 cells known to undergo a 50–70-fold increase in insulin binding while maintaining nearly constant levels of IGF-I binding during differentiation from preadipocytes into adipocytes. The presence of insulin/IGF receptor hybrids in 3T3-L1 adipocytes was demonstrated by the immunoprecipitation of phosphorylated receptors and a novel enzyme-linked immunoassay. Hybrid receptor levels were very low in the early stages of differentiation and increased rapidly between days 4 and 6, reaching a level about 100-fold higher in the mature adipocyte. Coincident with the hybrid assembly, the formation of archetypal (α2,β2) IGF receptors decreased. In fully differentiated adipocytes, virtually all of the IGF receptors were in hybrid form. Stimulation by IGF-I of receptors isolated from mature adipocytes caused autophosphorylation of IGF receptor β subunits in hybrid complexes, whereas autophosphorylated IGF holoreceptors were not demonstrable. Insulin and IGF-I were equipotent in stimulating glucose uptake in the differentiated adipocytes, leading to the conclusion that hybrid insulin/IGF receptors can transduce a transmembrane signal when activated by IGF-I. We conclude that hybrid formation constitutes a novel post-translational mechanism whereby increased synthesis of insulin receptors limits the cell surface expression of the homologous IGF receptor. Furthermore, biological actions in 3T3-L1 adipocytes, previously attributed to archetypal IGF receptors, are in fact mediated through hybrid receptors.

scholarly journals Comparison of insulin-like growth factor I receptor and insulin receptor purified from human placental membranes.

1986 ◽  
Vol 261 (35) ◽  
pp. 16727-16731
Author(s):  
Y Fujita-Yamaguchi ◽  
T R LeBon ◽  
M Tsubokawa ◽  
W Henzel ◽  
S Kathuria ◽  
...  
Keyword(s):  
Growth Factor ◽  
Insulin Receptor ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Placental Membranes ◽  
Growth Factor I
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