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High affinity binding sites for proinsulin in human IM-9 lymphoblasts

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Summary

Proinsulin and insulin binding in IM-9 lymphoblasts show curvilinear Scatchard plots, which may be explained by two binding sites, negative cooperativity 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.

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Abbreviations

αIR-3:

Monoclonal anti-IGF-1 receptor antibody

BSA:

bovine serum albumin

FACS:

fluorescence-activated cell sorting

FCS:

fetal calf serum

FITC:

fluorescein isothiocyanate

G1, S, G2 + M:

cell cycle phases

HEPES:

N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulphonic acid]

IGF-1/2:

insulin-like growth factor-1/2

MA-20:

monoclonal anti-insulin receptor antibody

NIDDM:

non-insulin-dependent diabetes mellitus

PBS:

phosphate buffered saline

TCA:

trichloroacetic acid

type 1:

IM-9 cell fraction with low insulin binding capacity

type 2:

IM-9 cell fraction with high insulin binding capacity

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Authors and Affiliations

  1. Abteilung Innere Medizin I, UniversitÄt Ulm, Robert-Koch-Strasse 8, D-89081, Ulm, Germany

    P. M. Jehle, M. P. Lutz &  Rolf D. Fussgaenger

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  1. P. M. Jehle
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  2. M. P. Lutz
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  3. Rolf D. Fussgaenger
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Jehle, P.M., Lutz, M.P. & Fussgaenger, R.D. High affinity binding sites for proinsulin in human IM-9 lymphoblasts. Diabetologia 39, 421–432 (1996). https://doi.org/10.1007/BF00400673

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  • DOI: https://doi.org/10.1007/BF00400673

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