Expression of annexins in the human neuroblastoma SH-SY5Y

Abstract

The annexins are a family of structurally related calcium dependent membrane-binding proteins [ I ] which can be shown to bind to phospholipid membranes in the presence of calcium [2].The annexins have been identified in a wide range of tissues from many species yet their physiological roles have not been clearly elucidated. They have been implicated in a variety of important cellular processes [3]. In particular annexin I1 seems to be a regulatory factor in the control of exocytosis [4,5].Annexin 11 is one of the best characterised components of the annexin family, occurring as a 36kD monomer and as a 90kDheterotetramer complex, containing two molecules of the 36kD heavy chain and two molecules of an 1 I k D light chain [6]. Evidence for the involvement of annexin 11 in the regulation of exocytosis is provided by the observations that the 36kD molecule can restore secretion in digitonin permeablised chromaffin cells [4] and that the annexin heterotetramer can restore secretion in streptolysin-0 permeabilised chromaffin cells, but must be phosphorylated by protein kinase C (PKC) to be effective [ 5 ] . The annexin 11 heavy chain is a major substrate for P K C and is phosphorylated at Ser-25 in the NH2 terminal region [7]. Annexin 11 has also been shown to promote the aggregation of chromaffin cell secretory granule membranes in a calcium dependent manner [8], and electron microscopic studies have shown that annexin 11 is concentrated between chromaffin granules and the plasma membrane in chromaffin cells [9].Previous studies by our group have shown that the human neuroblastoma SH-SY5Y expresses depolarisation and muscarinic M, receptor subtype evoked release of noradrenaline [lo]. Acute pretreatment with the PKC activating phorbol ester 12-0tetradecanoylphorbol-13-acetate (TPA) enhanced noradrenaline release [ I I], whereas addition of the selective PKC inhibitors R o 31 -7549 and Ro 31 -8220 inhibited noradrenaline release, indicating that PKC plays an important role in the release of noradrenaline from SH-SYSY.T h e first step in the investigation of the role of annexins in release of noradrenaline from SH-SY5Y was to determine which annexins were present in the cell. Annexins were isolated from SH-SYSY utilising the reversible calcium dependent binding properties of annexins to membrane phospholipids [ 121. These isolated annexins were separated on a two dimensional SDS PAGE gel and analysis of protein molecular weight and isoelectric points enabled the tentative identification of annexins l,ll,lV,V and VI by comparison with bovine lung annexins [12]. These results were confirmed by Western blot analysis of SH-SY5Y total homogenate. SH-SY5Y cells were harvested in Laemmli buffer and proteins separated by 12.5% SDS PAGE [I31 before transfer to polyvinylidene difluoride membranes. After blocking with 5% non fat milk solution, membranes were incubated with monoclonal antibodies to annexins I,II.IV and VI or with an affinity purified polyclonal antibody to annexin V. Immunolabelled proteins were identified by a p...