ABSTRACT: In AtT-20 cells ACTH secretion is regulated by both Ca2+ and G proteins. We previously demonstrated that calnuc, an EF-hand Ca2+ binding protein which regulates Alzheimer's beta-amyloid precursor protein (APP) biogenesis, binds both Ca2+ as well as G alpha subunits. Here we investigate calnuc's role in G protein-mediated regulation of ACTH secretion in AtT-20 neuroendocrine secretory cells stably overexpressing calnuc-GFP. Similar to endogenous calnuc, calnuc-GFP is mainly found in the Golgi, on the plasma membrane (PM), and associated with regulated secretion granules (RSG). By deconvolution immunofluorescence, calnuc-GFP partially colocalizes with G alpha i1/2 and G alpha i3 at the PM and on RSG. Cytosolic calnuc(DSS)-CFP with the signal sequence deleted also partially colocalizes with RSG and partially cosediments with G alpha i1/2 in fractions enriched in RSG. Overexpression of calnuc-GFP specifically increases the distribution of G alpha i1/2 on the PM whereas the distribution of G beta subunits and synaptobrevin 2 (Vamp 2) is unchanged. Overexpression of calnuc-GFP or cytosolic calnuc(DSS)-CFP enhances ACTH secretion two-fold triggered by mastoparan or GTPgamaS but does not affect glycosaminoglycan (GAG) chain secretion along the constitutive pathway or basal secretion of ACTH. Calnuc's facilitating effects on ACTH secretion are decreased after introducing anti-G alpha i1/2 , G alpha i3, G beta or calnuc IgG into permeabilized cells but not when G alpha 12 or preimmune IgG is introduced. The results suggest that calnuc binds to G alpha subunits on the Golgi and on RSG and that overexpression of calnuc causes redistribution of G alpha i subunits to the PM and RSG. Thus calnuc may connect G protein signaling and calcium signaling during regulated secretion.

Calnuc, a Golgi calcium binding protein, plays a key role in the constitution of calcium storage. Abnormal calcium homeostasis has been linked to Alzheimer's disease (AD). Excessive production and/or accumulation of beta-amyloid (Abeta) peptides that are proteolytically derived from the beta-amyloid precursor protein (APP) have been linked to the pathogenesis of AD. APP has also been indicated to play multiple physiological functions. In this study, we demonstrate that calnuc interacts with APP through direct binding to the carboxyl-terminal region of APP, possibly in a calcium-sensitive manner. Immunofluorescence study revealed that the two proteins co-localize in the Golgi in both cultured cells and mouse brains. Over-expression of calnuc in neuroblastoma cells significantly reduces the level of endogenous APP. Conversely, down-regulation of calnuc by siRNA increases cellular levels of APP. Additionally, we show that over-expression of calnuc down-regulates the APP mRNA level and inhibits APP biosynthesis, which in turn results in a parallel reduction of APP proteolytic metabolites, sAPP, CTFs and Abeta. Furthermore, we found that the level of calnuc was significantly decreased in the brain of AD patients as compared with that of age-matched non-AD controls. Our results suggest a novel function of calnuc in modulating the levels of APP and its proteolytic metabolites, which may further affect the patho/physiological functions of APP including AD pathogenesis.