DIFFERENTIAL CELLULAR DYNAMICS OF INSULIN RECEPTOR A AND B.

GIUDICE JIMENA; COLUCCIO LESKOW FEDERICO; ARNDT-JOVIN DONNA M; JOVIN THOMAS M; JARES-ERIJMAN ELIZABETH

Buenos Aires. Argentina

Jornada; XI Jornadas Anuales de las Sociedad Argentina de Biología; 2009

Sociedad Argentina de Biología

<!-- /* Font Definitions */ @font-face {font-family:SimSun; panose-1:2 1 6 0 3 1 1 1 1 1; mso-font-alt:宋体; mso-font-charset:134; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:3 135135232 16 0 262145 0;} @font-face {font-family:"@SimSun"; panose-1:2 1 6 0 3 1 1 1 1 1; mso-font-charset:134; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:3 135135232 16 0 262145 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:SimSun;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Insulin signalling comprises a complex cascade of events playing a key role in the regulation of glucose metabolism and cellular growth. Failures in its function lead to diabetes and disregulation of its signalling pathway was described in many cancer models. Insulin receptor (IR) is a tetrameric receptor tyrosine kinase. Two splice variants exist in mammalian cells: IR-A lacking exon 11, and the full length IR-B. Aim and methods. In order to study the dynamics of the activation and internalization of the IR isoforms by microscopy in living cells we combined 2 powerful labelling techniques: streptavidin-QDs conjugated with biotinylated ligands; and visible fluorescent proteins (VFPs). Results and discussion. We generated IR fused to eGFP, CFP and YFP without affecting functionality. We demonstrated that biotin amido caproyl bovine insulin was able to bind IR and IR-VFPs (A and B) in living cells, to activate it and to induce internalization which could be imaged by confocal and programmable array microscopy (PAM). Determination of IR internalization levels in a cell by cell basis showed an early differential rate for IR-A-GFP (65±11%) and for IR-B-GFP (47±13%). These tools will allow us to study interactions between insulin and IR, binding, endocytosis and recycling processes.