CONICET | Buscador de Institutos y Recursos Humanos

Insulin regulates a variety of cellular processes such as transport and metabolism of glucose, lipids and proteins, nucleic acid synthesis and expression of certain genes. Two splice variants of insulin receptor (IR) exist in mammalian cells: IR-A lacking 12 aminoacids coded by exon 11, and the full length IR-B. Both isoforms behave differently in the presence of ligands and cannot be distinguished by antibodies. Aim and methods. In order to study the dynamics of the IR in living cells we applied a labeling scheme based on the covalent modification of cell surface proteins making use of the post-translational modification of the acyl carrier protein by a phosphopantetheinyl transferase (ACPwt-S) which transfers the 4-phosphopantetheine from coenzyme A (CoA) to a conserved serine residue of ACP. Results and discussion. We cloned the IR-A and IR-B with an small tag (12 or 36 aminoacids) inserted in the position 1878 (pair of bases) of IR. In this way we could label extracellular domain of IR in living cells for the first time using ACPwt-S and fluorescent or biotinylated CoA. In addition, these chimeras bind biotinylated insulin revealed by confocal microscopy using streptavidin quantum dots or fluorescent streptavidin.