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

Area of Science:

• Endocrinology and Metabolism
• Cell Biology
• Molecular Signaling

Background:

• Insulin and proinsulin binding to IM-9 lymphoblasts typically show complex Scatchard plots, suggesting multiple receptor populations or cooperativity.
• Previous studies indicated heterogeneity in insulin receptor expression and function within cell lines.

Purpose of the Study:

• To investigate the heterogeneity of insulin and proinsulin binding sites in IM-9 lymphoblasts.
• To characterize distinct cell populations based on their receptor binding properties and signaling responses.

Main Methods:

• Flow-cytometric analysis was employed to separate IM-9 lymphoblasts into distinct cell fractions based on insulin binding.
• Radioligand binding assays (using 125I-insulin and 125I-proinsulin) were performed on separated cell fractions to determine binding affinity (Kd) and capacity (sites/cell).
• Competitive binding studies using specific antibodies (MA-20 for insulin receptor, alpha IR-3 for IGF-1 receptor), unlabeled ligands, and C-peptide were conducted.
• Insulin-induced tyrosine phosphorylation and cell cycle analysis (DNA analysis) were assessed.

Main Results:

• Two distinct cell populations (Type 1 and Type 2) were identified: Type 1 cells exhibited low-capacity, high-affinity insulin binding, characteristic of typical insulin receptors, with limited proinsulin competition.
• Type 2 cells displayed high-capacity, lower-affinity insulin binding but showed significantly higher affinity for proinsulin binding, which was inhibited by C-peptide but not the insulin receptor antibody MA-20.
• Proinsulin binding to Type 2 cells was resistant to insulin-induced downregulation and activated distinct signaling cascades (greater tyrosine phosphorylation of a 65 kDa protein) compared to insulin.
• Type 1 cells were predominantly in G1 phase, while Type 2 cells were in S and G2+M phases of the cell cycle.

Conclusions:

• IM-9 lymphoblasts can be separated into fractions with distinct receptor populations: typical insulin receptors and high-affinity proinsulin binding sites.
• These proinsulin binding sites differ from classical insulin receptors in affinity, ligand specificity (C-peptide inhibition), kinetics, resistance to downregulation, and signaling pathways.
• The observed differences in receptor populations and cell cycle distribution suggest functional heterogeneity within the IM-9 lymphoblast cell line.