Binding and signaling of insulin glargine and its metabolites to the human insulin receptor isoform A and B.

<p>Binding of the insulin analogs to the human IR-A (<b>A</b>) or IR-B (<b>B</b>) was analyzed in a competitive binding assay using SPA technology. The binding of a constant concentration of [¹²⁵I]insulin to plasma membranes from CHO cells overexpressing either IR-A or IR-B was measured in presence of increasing concentrations of unlabeled competing ligand after incubation at room temperature for 12 h. All data has been corrected for non-specific binding and are expressed as percentage of [¹²⁵I]insulin in absence of competing ligand. To analyze the insulin-stimulated activation and subsequent autophosphorylation of the insulin receptor CHO cells overexpressing the human IR-A (<b>C</b>) or IR-B (<b>D</b>) were stimulated for 15 min at 37°C with increasing concentrations of peptides, then the cells were fixed with 3.7% PFA and the amount of phosphotyrosines was analyzed via In-Cell Western. The data represent mean values ± SEM of at least 3 individual experiments measured in quadruplicate.