The mammalian Sec6/8 complex interacts with Ca2+ signaling complexes and regulates their activity

The localization of various Ca2+ transport and signaling proteins in secret ory cells is highly restricted. resulting in polarized agonist-stimulated C a2+ waves. In the present work, we examined the possible roles of the Sec6/ 8 complex or the exocyst in polarized Ca2+ signaling in pancreatic acinar c ells. Immunolocalization by confocal microscopy showed that the Sec6/8 comp lex is excluded from tight junctions and secretory granules in these cells. The Sec6/8 complex was found in at least two cellular compartments, part o f the complex showed similar, but not identical, localization with the Golg i apparatus and part of the complex associated with Ca2+ signaling proteins next to the plasma membrane at the apical pole. Accordingly, immunoprecipi tation (IP) of Sec8 did not coimmunoprecipitate beta COP, Golgi 58K protein , or mannosidase II, all Golgi-resident proteins, By contrast, IP of Sec8 c oimmunoprecipitates Sec6, type 3 inositol 1,4,5-trisphosphate receptors (IP (3)R3), and the G beta gamma subunit of G proteins from pancreatic acinar c ell extracts. Furthermore, the anti-Sec8 antibodies coimmunoprecipitate act in, Sec6, the plasma membrane Ca2+ pump, the G protein subunits G alpha q a nd G beta gamma, the beta 1 isoform of phospholipase C, and the ER resident IP(3)R1 from brain microsomal extracts. Antibodies against the various sig naling and Ca2+ transport proteins coimmunoprecipitate Sec8 and the other s ignaling proteins. Dissociation of actin filaments in the immunoprecipitate had no effect on the interaction between Sec6 and Sec8, but released the a ctin and dissociated the interaction between the Sec6/8 complex and Ca2+ si gnaling proteins. Hence, the interaction between the Sec6/8 and Ca2+ signal ing complexes is likely mediated by the actin cytoskeleton. The anti-Sec6 a nd anti-Sec8 antibodies inhibited Ca2+ signaling at a step up-stream of Ca2 + release by IP3. Disruption of the actin cytoskeleton with latrunculin B i n intact cells resulted in partial translocation of Sec6 and Sec8 from memb ranes to the cytosol and interfered with propagation of agonist-evoked Ca2 waves. Our results suggest that the Sec6/8 complex has multiple roles in s ecretory cells including governing the polarized expression of Ca2+ signali ng complexes and regulation of their activity.