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

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, alpha IR-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 alpha IR-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.