The ICA512 extracellular domain: unraveling its role in cell adhesion and signaling in pancreatic beta-cells.

JUHA M. TORKKO; MARÍA E. PRIMO; ELISA VERGARI; RONALD DIRKX; ANTJE VIEHRIG; MARIO R. ERMÁCORA; MICHELE SOLIMENA

Dresden

Congreso; 3rd International Congress on Stem Cells and Tissue Formation; 2010

DFG-Research Center for Regenerative Therapies Dresden (CRTD) and the Collaborative Research Center (SFB 655) "Cells into Tissues"

ICA512/IA-2/RPTPN is a catalytically inactive receptor tyrosine phosphatase enriched in the membrane of insulin granules (ISGs) of pancreatic beta-cells. ICA512 null mice display impaired glucose tolerance and insulin secretion. We previously showed that upon ISG exocytosis ICA512 is transiently inserted into the plasma membrane, where its cytoplasmic tail is cleaved by calpain-1. The resulting cytosolic fragment promotes insulin output by mobilizing ISGs from the cortical cytoskeleton as well as by promoting ISG biogenesis and beta-cell proliferation by enhancing the activity of STAT5 and STAT3. Structural studies have further revealed that its mature ectodomain (ME-ICA512) includes the SEA domain of mucins and can form homoand heterodimers. Like mucins, ME-ICA512 displays self-proteolytic activity in vitro. Here we report that a GFP-tagged ICA512 construct lacking the entire N-terminal extracellular region except for the ME-ICA512 domain (delta-NTF-ICA512-GFP) is constitutively targeted to the cell surface of insulinoma (INS-1) cells, but does not appear to be cleaved by calpain upon stimulation. We obtained a monoclonal antibody that only reacts with ME-ICA512 when N-glycosylation at N506 of the latter is prevented. Using this antibody for studies of recombinant variants of ME-ICA512 in vitro and of delta-NTF-ICA512-GFP in INS-1 cells, we have gathered evidence suggesting that meIA-2, similar to mucins, may undergo O-glycosylation. N-terminal microsequencing of delta-NTF-ICA512-GFP from INS-1 cells further points to the possibility that ME-ICA512 undergoes proteolysis in vivo. Taken together, our studies provide mechanistic insight into how the ICA512 extracellular domain could participate in cell adhesion as well as in regulation of insulin secretion and beta-cell proliferation by modulating the intracellular signaling of this receptor.