Studies on the role of annexin II in rat basophils

The annexins are a family of calcium-dependent phospholipid-binding proteins. They are defined by the presence of a highly conserved 70 amino acid domain responsible for binding calcium ions and interacting with phospholipid membranes. All annexins contain four copies of this repeat except for annexin VI, which has eight. In addition, each annexin has a unique N-terminus, varying in size from around 10 to nearly 200 amino acids. Ten different annexins have been cloned in mammals and a further three in lower eukaryotic phyla. No functions have been unequivocally assigned to any members of the family. Most annexins have a restricted tissue distribution, but adl cells express at least one member of the family. A secretory cell line, rat basophilic leukaemia RBL-2H3 cells was shown to express annexins I, II, VI and VII, all of which have been implicated in exocytosis. Immunolabelling of annexin II in RBL-2H3 cells, revealed that it is localised to the cortical region and to punctate bodies in the cytosol. The presence of annexin II close to the plasma membrane, and possibly on secretory vesicles located in the cytosol, is consistent with a role in exocytosis. Several rat annexin II cDNA clones were isolated using a murine annexin II cDNA to screen a plasmid cDNA library. One of these was fully sequenced. Alignment of the rat annexin II cDNA sequence with those from other species revealed a 6-nucleotide insert in the coding region of one of the clones, close to the N-terminus. The insertion of 2 amino acids at this point may have important functional consequences since it is close to the sites of phosphorylation and the region that interacts with annexin II's cellular ligand, pll. To demonstrate the existence of this insert in vivo, primer extension analysis was performed on mRNA extracted from RBL-2H3 cells. To investigate the effect on secretion of a reduction in annexin II expression, RBL-2H3 cells were transfected with an expression plasmid containing rat annexin II cDNA in reverse orientation. A clone expressing significantly lower than wild type levels of annexin II was shown to have reduced secretory response to Ca2+ and GTPγS. Over a period of weeks this clone reverted to wild type levels of annexin II expression, and this coincided with a return to normal secretory response. The results in this thesis support the theory that annexin II functions in the secretory pathway