In:
The Journal of Cell Biology, Rockefeller University Press, Vol. 150, No. 5 ( 2000-09-04), p. 1101-1112
Kurzfassung:
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 βCOP, 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 Gβγ 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 Gαq and Gβγ, the β1 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.
Materialart:
Online-Ressource
ISSN:
0021-9525
,
1540-8140
DOI:
10.1083/jcb.150.5.1101
Sprache:
Englisch
Verlag:
Rockefeller University Press
Publikationsdatum:
2000
ZDB Id:
1421310-2
SSG:
12
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