UC San Diego

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.