Original Article

The Mammalian Sec6/8 Complex Interacts with Ca²⁺ Signaling Complexes and Regulates their Activity

Correspondence to: Shmuel Muallem, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-9040. Tel:(214) 648-2593 Fax:(214) 648-8879 E-mail:shmuel.muallem{at}email.swmed.edu.

The localization of various Ca²⁺ transport and signaling proteins in secretory cells is highly restricted, resulting in polarized agonist-stimulated Ca²⁺ 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 cells. Immunolocalization by confocal microscopy showed that the Sec6/8 complex is excluded from tight junctions and secretory granules in these cells. The Sec6/8 complex was found in at least two cellular compartments, part of the complex showed similar, but not identical, localization with the Golgi apparatus and part of the complex associated with Ca²⁺ signaling proteins next to the plasma membrane at the apical pole. Accordingly, immunoprecipitation (IP) of Sec8 did not coimmunoprecipitate ßCOP, Golgi 58K protein, or mannosidase II, all Golgi-resident proteins. By contrast, IP of Sec8 coimmunoprecipitates Sec6, type 3 inositol 1,4,5-trisphosphate receptors (IP₃R3), and the Gß subunit of G proteins from pancreatic acinar cell extracts. Furthermore, the anti-Sec8 antibodies coimmunoprecipitate actin, Sec6, the plasma membrane Ca²⁺ pump, the G protein subunits Gq and Gß, the ß1 isoform of phospholipase C, and the ER resident IP₃R1 from brain microsomal extracts. Antibodies against the various signaling and Ca²⁺ transport proteins coimmunoprecipitate Sec8 and the other signaling proteins. Dissociation of actin filaments in the immunoprecipitate had no effect on the interaction between Sec6 and Sec8, but released the actin and dissociated the interaction between the Sec6/8 complex and Ca²⁺ signaling proteins. Hence, the interaction between the Sec6/8 and Ca²⁺ signaling complexes is likely mediated by the actin cytoskeleton. The anti-Sec6 and anti-Sec8 antibodies inhibited Ca²⁺ signaling at a step upstream of Ca²⁺ release by IP₃. Disruption of the actin cytoskeleton with latrunculin B in intact cells resulted in partial translocation of Sec6 and Sec8 from membranes to the cytosol and interfered with propagation of agonist-evoked Ca²⁺ waves. Our results suggest that the Sec6/8 complex has multiple roles in secretory cells including governing the polarized expression of Ca²⁺ signaling complexes and regulation of their activity.

Key Words: Sec6/8 complex, Ca²⁺ signaling proteins, assembly, actin cytoskeleton, Ca²⁺ signaling

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