The role of Rab3D in pancreatic acinar secretion.
Chen, Xuequn
2003
Abstract
Rab3 proteins are believed to play an important role in regulated exocytosis and previous work has demonstrated the presence of Rab3D on pancreatic zymogen granules. To further understand the function of Rab3D in acinar exocytosis, adenoviral constructs were prepared encoding HA-tagged wild type Rab3D and three mutant forms-N135I and T36N (dominant negative) and Q81L (constitutive active) which also expressed EGFP driven by a separate promoter. The adenoviral infection efficiency was nearly 100% as visualized by expression of EGFP. Control beta-Gal virus infection had no effect on either basal or CCK-induced secretion. While the expression of Rab3D and Rab3D Q81L had no effect, Rab3D N1351 and T36N functioned as dominant negative mutants and inhibited CCK-induced amylase release by 40--50% at all points on the CCK dose-response curve from 3--300 pM. Inhibition was stronger during the first 5 minutes (71 +/- 5%) than over 30 minutes (36% +/- 5%). Similar inhibition was found using other agonists including bombesin, carbachol, A23187 and cAMP. Localization of adenoviral expressed Rab protein showed wild type Rab3D localized to zymogen granules. The two dominant negative mutants did not localize to granules and were primarily in the basolateral region of the cell. These results suggest that Rab3D plays an important role in regulating the terminal steps of acinar exocytosis and that this effect is preferentially on the early phase of amylase release. To evaluate the potential mechanisms by which the dominant negative mutants might act, an affinity-precipitation assay based on the property of the Rab3 effector Rim1 to interact only with GTP bound Rab3D was developed. Using acinar lysate loaded with GDP and GTPgammaS as negative and positive controls, the percentage of endogenous Rab3D in the GTP bound form was found to be 78.9% +/- 4.5% and almost all GTP bound Rab3D was membrane associated. Overexpression of HA-tagged Rab3D, and its Q81L, N135I and T36N mutants had no effect on the total amount of endogenous RAb3D. However, both dominant negative mutants, T36N and N135I, reduced GTP bound endogenous Rab3D by about 70%, while the wild type and Q81L had no effect. Triton X-114 phase separation and cell fractionation studies showed that dominant negative Rab3D mutants did not alter isoprenylation or membrane association of endogenous RAb3D. Zymogen granules were purified and the dominant negative Rab3D was found not to affect the amount of endogenous Rab3D on the granules revealed by either Western blotting or immunocytochemistry. Dominant negative Rab3D, however, reduced the GTP bound form of endogenous Rab3D by about 80%. The two dominant negative Rab3D mutants, therefore, interfere with endogenous Rab3D function by blocking the GDP/GTP exchange but not zymogen granule targeting of endogenous Rab3D.Subjects
Exocytosis Pancreatic Acinar Rab3d Role Secretion
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