ISSN:
1432-0428
Keywords:
Keywords Insulin receptor heterogeneity
;
proinsulin binding
;
C-peptide binding
;
signal transduction.
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
Summary Proinsulin and insulin binding in IM-9 lymphoblasts show curvilinear Scatchard plots, which may be explained by two binding sites, negative co-operativity of receptors, or both. Using flow-cytometric analysis of insulin binding, we were able to distinguish and separate two different IM-9 cell fractions. In both fractions, Scatchard plots for specific binding of insulin and proinsulin were linear, suggesting the presence of two distinct populations of receptors. Type 1 cells showed low capacity but high affinity of insulin binding (16,300 ± 3,000 sites/cell; Kd 0.4 ± 0.1 nmol/l). Proinsulin and insulin-like growth factor 1 (IGF-1) were significantly less potent in competition. MA-20, a specific antibody against human insulin receptors, inhibited insulin binding by 80 %, while the specific antibody against human IGF-1 receptors, αIR-3, had no effect. Pretreatment with insulin decreased insulin binding by 90 %. 125I-insulin displayed stepwise dissociation with the rate markedly enhanced by cold insulin. Type 2 cells exhibited significantly different binding characteristics with higher capacity but lower affinity of 125I-insulin binding (430,000 ± 25,000 sites/cell, p 〈 0.001 vs type 1; Kd 2 ± 0.4 nmol/l, p 〈 0.02 vs type 1). Proinsulin competed with similar potency for insulin binding, while IGF-1 was still less potent. 125I-proinsulin showed a significantly higher binding affinity than 125I-insulin (Kd 0.5 ± 0.3 nmol/l, p 〈 0.05) with 50,000 ± 10,000 binding sites/cell. C-peptide was able to compete for 125I-proinsulin, but not for 125I-insulin binding. MA-20 did not influence 125I-proinsulin binding, but inhibited 125I-insulin binding by 50 %, whereas αIR-3 increased proinsulin binding 1.5-fold with no effect on insulin binding. Preincubation with insulin decreased insulin binding by 50 % and proinsulin binding by 10 %. The dissociation of 125I-proinsulin showed linear first-order kinetics and was not significantly accelerated by cold proinsulin. Furthermore, the tyrosine phosphorylation of a 65 kDa protein was stimulated to a significantly greater extent by proinsulin than by insulin, indicating activation of different signalling cascades. DNA analysis revealed that type 1 cells were predominantly in the G1 phase, whereas type 2 cells were in the S and G2 + M phases of the cell cycle. We conclude, that IM-9 lymphoblasts were separated by flow-cytometry into one fraction with typical insulin receptors and a second fraction with high affinity binding sites for proinsulin. High affinity proinsulin binding sites were distinguished from typical insulin receptors by: 1) higher affinity for proinsulin than insulin, 2) inhibition of proinsulin binding by C-peptide but not by the insulin receptor antibody MA-20, 3) non-co-operative first order dissociation kinetics of proinsulin binding, 4) resistance to down-regulation by insulin, and 5) differences in signal transduction. [Diabetologia (1996) 39: 421–432]
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00400673
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