NEW INSIGHTS ON ICA512 AND INSULIN INTERACTION

ERMÁCORA, MARIO R.; LLOVERA, RAMIRO E.; MÜLLER ANDREAS; TOLEDO, PAMELA L.; SOLIMENA, MICHELE; TORKKO, JUHA M.

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

ICA512/IA?2/PTPRN is a decoy receptor protein tyrosine phosphatase enriched in secretory granules (SGs) of the pancreatic β-cells and other neuroendocrine cells. Previous studies imply its involvement in the biogenesis, trafficking and exocytosis of insulin SGs, as well as in β-cell proliferation. Its regulated endocrine-specific protein 18 homologous domain (RESP18HD) is necessary and sufficient for the sorting of ICA512 to SGs of rat insulinoma INS-1 cells and its binding with high-affinity to insulin and proinsulin. In previous studies we observed the formation of complexes between insulin and RESP18HD 35-131 by co-aggregation in vitro. We now show by EM negative staining that complexes of insulin and RESP18HD are amorphous, in contrast to RESP18HD alone, which instead self-aggregates in a moderately ordered fashion. ICA512-RESP18HD consists at least two distinct regions, including a N-terminal Cys-rich motif (aa 35-90) and an intrinsically disordered region in the C-terminal part of the domain (aa 91-131). Fluorescence microscopy of RESP18HD-TQ2 tagged fusion protein variants in INS-1 cells shows that the region encompassing the signal peptide (aa 1-34) followed by the Cys-rich motif of the RESP18HD (aa 35-90) contains sufficient information for SG targeting. Moreover, co-expression of different RESP18HD-TQ2 variants with Insulin-Venus fusion to assess Fluorescence lifetime imaging-fluorescence resonance energy transfer (FLIM-FRET), revealed moderate interaction (FRET) of these proteins also in the cells. These results further underscore the enabling role of ICA512-RESP18HD in targeting insulin and possibly other cargoes to the SGs.