Endocrine Abstracts

Annexin 1 (ANXA1) is a 37 Kd member of the annexin family of Ca²⁺− and phospholipid binding proteins that has been well demonstrated to act as a mediator of glucocorticoid action in inflammation and in the control of anterior pituitary hormone release. ANXA1 is also proapoptotic and has been identified as a mediator of apoptotic cell engulfment (Solito et al. 2003 FASEB J 17:1544; Arur et al. 2003 Dev Cell 4:587). Using yeast two hybrid screening we have investigated binding partners of annexin 1. A fusion bait encoding full length mouse ANXA1 was used to screen a mouse brain two-hybrid cDNA library. Interacting clones were selected under stringent conditions by growth on quadruple drop-out plates and X-gal overlay assay. 87 clones displayed ANXA1-dependent transactivation of yeast reporter genes and were subjected to sequencing and database searches to identify the corresponding proteins. Proteins/genes identified included the protease calpain (capns1), the tyrosine kinase interacting protein RanBP9, the cdk1 inhibitory kinase myt 1, the cell adhesion molecule contactin 4/BIG-2, and the breast cancer associated gene BRCA1. As calpain has been associated with glucocorticoid-induced apoptotic signalling we went on to investigate the calpain interaction further. Immunofluorescence studies confirmed that ANXA1 and calpain co-localise in folliculo-stellate-like TtT/GF cells and pituitary folliculo-stellate cells. Western blot analysis of ANXA1 protein in TtT/GF cells treated with cycloheximide (to induce apoptosis) revealed that cycloheximide (50 μM, 24 h) stimulated an increase in the amount of N terminal cleaved ANXA1 (32 Kd) which was abolished by co-treatment with the calpain inhibitors, calpain inhibitor II (1 μM, 24 h) or PD150606 (1 μM, 24 h). These results indicate that ANXA1 and calpain associate in vivo and suggest that proteolytic cleavage of ANXA1 is involved in apoptotic signalling.