Figure 2. Effect of insulin, glargine and IGF1 on PIP₃ production in intact living cells.

Activation of tyrosine kinase receptors by their ligands stimulates the activity of PI-3 kinase, leading to increased phosphorylation of phosphatidyl-inositol 2 phosphate (PIP₂) into phosphatidyl-inositol 3 phosphate (PIP₃) and subsequent recruitment of Akt to the plasma membrane through its pleckstrin homology (PH) domain. To monitor the production of PIP₃ induced by receptor activation, cells were co-transfected with cDNAs coding for the PH domain of Akt fused to luciferase (Luc-Akt-PH) and YFP fused to the membrane localization sequence of neuromodulin. Cells were pre-incubated for 10 min with coelenterazine and then stimulated with increasing ligand concentrations. (A) Typical experiment showing real-time insulin or IGF1 effects on PIP₃ production in MCF-7 cells. (B) Dose-dependent effect of insulin, glargine and IGF1 on PIP₃ production in MCF-7 and MDA-MB231 cells. Ligand-induced BRET (BRET above basal at the plateau) was determined for each ligand concentration and was used to establish dose-response curves. Results are the means ± S.E.M. of 5 to 6 independent experiments. EC50 for insulin, IGF1, glargine and other insulin analogues are given in Table 2. (C) Left panel: Receptors were partially purified from MDA-MB231 and MCF-7 cells by WGL chromatography. WGL eluates (12 µg of protein) were submitted to electrophoresis and western-blotting. IR and IGF1R expression levels were evaluated by immunoblotting (IB) using anti-IR (C-19) and anti-IGF1R (C-20) antibodies. Right panel: After normalization of the eluates for IR content, immunoprecipitation (IP) was performed using anti-IR antibody (CT1) and hybrid receptors were detected using anti-IGF1R antibody. Blots were then stripped and reprobed with anti-IR antibody. Results are representative of 6 immunoprecipitation experiments performed on three independent batches of receptor preparations (**, p<0.01).