Membrane Association Domains in Ca2+-dependent Activator Protein for Secretion Mediate Plasma Membrane and Dense-core Vesicle Binding Required for Ca2+-dependent Exocytosis

Abstract

Ca2؉ -dependent activator protein for secretion (CAPS) is a cytosolic protein essential for the Ca 2؉ -dependent fusion of dense-core vesicles (DCVs) with the plasma membrane and the regulated secretion of a subset of neurotransmitters. The mechanism by which CAPS functions in exocytosis and the means by which it associates with target membranes are unknown. We identified two domains in CAPS with distinct membrane-binding properties that were each essential for CAPS activity in regulated exocytosis. The first of these, a centrally located pleckstrin homology domain, exhibited three properties: charge-based binding to acidic phospholipids, binding to plasma membrane but not DCV membrane, and stereoselective binding to phosphatidylinositol 4,5-bisphosphate. Mutagenesis studies revealed that the former two properties but not the latter were essential for CAPS function. The central pleckstrin homology domain may mediate transient CAPS interactions with the plasma membrane during Ca 2؉ -triggered exocytosis. The second membrane association domain comprising distal C-terminal sequences mediated CAPS targeting to and association with neuroendocrine DCVs. The CAPS C-terminal domain was also essential for optimal activity in regulated exocytosis. The presence of two membrane association domains with distinct binding specificities may enable CAPS to bind both target membranes to facilitate DCV-plasma membrane fusion.

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation

“…However, Ser-1281 phosphorylation may regulate other aspects of CAPS function. C-terminal sequences in CAPS are essential for interactions with dense-core vesicles (19). These could mediate the reported CAPS stimulation of catecholamine loading by vesicular amine transporter in chromaffin cells (35).…”

“…In addition to the pleckstrin homology and C2 domains that have been identified as essential for CAPS function (5,19,51), this study characterizes a functionally important N-terminal phosphorylation domain in CAPS.…”

“…Preliminary in vitro studies with purified phosphatases showed that the N-terminal phosphorylation sites of CAPS were resistant to dephosphorylation by phosphatases PP1, PP2A, and PP2B/calcineurin. 3 Phosphatidylinositol 4,5-diphosphate is essential for priming evoked vesicle exocytosis (50) and serves as a co-factor for CAPS membrane recruitment and activation (4,19,51). Protein kinase CK2 was reported to be inhibited by the binding of phosphatidylinositol 4,5-diphosphate (52).…”

“…Cell Labeling, Transfection, and Immunoprecipitation-PC12 cells and COS-1 cells were cultured and transfected by electroporation (18,19). Cells were incubated in phosphatefree Dulbecco's modified Eagle's medium (Sigma) for 30 min and labeled with 1 mCi of [ 32 P]orthophosphate (GE Healthcare) for 1 h. For CK2 inhibition, 60 M 4,5,6,7-tetrabromobenzotriazole (Calbiochem) was added to the labeling medium.…”

Interaction of Calcium-dependent Activator Protein for Secretion 1 (CAPS1) with the Class II ADP-ribosylation Factor Small GTPases Is Required for Dense-core Vesicle Trafficking in the trans-Golgi Network

“…On the other hand, recent studies using knock-out (KO) or knockdown (KD) approaches have led to fresh debates concerning the involvement of CAPS1 in the priming of synaptic vesicle exocytosis (10) as well as in the vesicular loading of catecholamine or serotonin (11,12). Although these previous studies focused on the role of CAPS protein in exocytosis at secretion sites, such as nerve terminals or the cell periphery, a large fraction of CAPS protein in many neuronal cell types is actually localized in the soma rather than at these secretion sites (2,13,14). Therefore, the role of somal CAPS proteins, which constitute the largest amount of CAPS proteins in the cell, also needs to be elucidated.…”