Subcellular Distribution and Function of Rab3A-D in Pancreatic Acinar AR42J Cells

Piiper, Albrecht; Leser, Jürgen; Lutz, Manfred P.; Beil, Michael; Zeuzem, Stefan

doi:10.1006/bbrc.2001.5651

Cited by 24 publications

(23 citation statements)

References 35 publications

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“…Rab3A, Rab3B, and Rab3C are expressed primarily in the brain and endocrine cells, whereas Rab3D is synthesized in a different pattern with the highest levels in exocrine cells and adipose tissue. These results extend previous data (6,7,12,43) by quantifying the concentrations of various Rab3 proteins. As a result, we show for example that the concentration of Rab3D in the lachrymal and parotid glands is twice that of Rab3A in the brain (where Rab3A is the most abundant Rab protein (2)) and that most tissues do not express detectable levels of any Rab3 protein (Fig.…”

Section: Discussionsupporting

confidence: 90%

“…In this regard, Rab3 proteins are similar to Rab11b, which we recently found to be the only Rab protein tested among multiple such proteins that inhibited exocytosis. 2 Overall, the available data reported in this and other studies (12,14,20) indicate that Rab3A, Rab3B, Rab3C, and Rab3D are functionally comparable based on similar localizations, a high degree of sequence homology, and equivalent inhibition of exocytosis in transfected PC12 cells. Clearly there are major differences in the cells and tissues that express the different Rab3 isoforms and possibly also in the functions of Rab3 proteins, because the biogenesis and fusion of secretory vesicles in exocrine glands containing Rab3D exhibit quite different properties from the biogenesis and fusion of endocrine and neuronal secretory vesicles.…”

Section: Discussionsupporting

confidence: 55%

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Localization Versus Function of Rab3 Proteins

Schlüter

Khvotchev

Jahn

et al. 2002

Journal of Biological Chemistry

149

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Rab3A, Rab3B, Rab3C, and Rab3D constitute a family of GTP-binding proteins that are implicated in regulated exocytosis. Various localizations and distinct functions have been proposed for different and occasionally even for the same Rab3 protein. This is exemplified by studies demonstrating that deletion of Rab3A in knock-out mice results in dysregulation of the final stages of exocytosis, whereas overexpression of Rab3A in neuroendocrine cells causes nearly complete inhibition of Ca 2؉-triggered exocytosis. We have now examined the properties of all Rab3 proteins in the same assays, with the long-term goal of identifying a common conceptual framework for their functions. Using quantitative immunoblotting, we found that all four Rab3 proteins were expressed in brain and endocrine tissues, although at widely different levels. Rab3A, Rab3B, and Rab3C co-localized to synaptic and secretory vesicles consistent with potential redundancy, whereas Rab3D was expressed at high levels only in the endocrine pituitary (where it was more abundant than Rab3A, Rab3B, and Rab3C combined), in exocrine glands, and in adipose tissue. In transfected PC12 cells, all four Rab3 proteins strongly inhibited Ca 2؉ -triggered exocytosis. Except for a mutation that fixes Rab3 into a permanently GDP-bound state, all Rab3 mutations tested had no effect on this inhibition, including a mutation in the calmodulin-binding site that was described as inactivating (Coppola, T., Perret-Menoud, V., Lü thi, S., Farnsworth, C. C., Glomset, J. A., and Regazzi, R. (1999) EMBO J. 18, 5885-5891). Unexpectedly, overexpression of wild type Rab3A and permanently GTP-bound mutant Rab3A in PC12 cells caused a loss of secretory vesicles and an increase in constitutive, Ca 2؉ -independent exocytosis that correlated with the inhibition of regulated Ca 2؉ -triggered exocytosis. Our data indicate that overexpression of Rab3 in PC12 cells impairs the normal control of the final step in exocytosis, thereby converting the regulated secretory pathway into a constitutive pathway. These results offer an hypothesis that reconciles Rab3 transfection and knockout studies by suggesting that Rab3 functions as a gatekeeper of a late stage in exocytosis.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionsupporting

confidence: 55%

Localization Versus Function of Rab3 Proteins

Schlüter

Khvotchev

Jahn

et al. 2002

Journal of Biological Chemistry

149

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“…We and others have previously demonstrated that Rab3D is expressed in AR42J cells and that Rab3D levels increase upon treatment with Dex [23,28,30], suggesting that Rab3D plays a role in regulated exocytosis in these cells. It should also be noted that the other three Rab3 isoforms (Rab3A, Rab3B and Rab3C) are expressed in this cell line [23,28]. Interestingly, the four Rab3 isoforms do not co-localize with one another [23], suggesting that they may have distinct roles in these cells.…”

Section: Expression and Subcellular Localization Of Rab3d In Ar42j Cellsmentioning

confidence: 66%

“…Because of its localization on the ZG membrane, Rab3D was originally believed to be involved in late events in exocytosis. However, overexpression of Rab3D and Rab3D mutants does not affect regulated secretion in similar ways in all cell types examined [22][23][24]. Moreover, in Rab3D knockout mice, granule size, but not agonist-induced secretion, was altered in pancreatic and parotid acinar cells [25].…”

Section: Introductionmentioning

confidence: 91%

“…CCK-8 induces amylase release from AR42J cells and, therefore, this cell line represents a convenient model for studying the molecular events involved in regulated secretion in exocrine cells. Rab3D, as well as the other three Rab3 isoforms (A, B and C), are all expressed in AR42J cells [23,28]. Moreover, Rab3D is upregulated in response to Dex treatment, suggesting a role for this protein in AR42J cells as they develop a more acinar cell-like phenotype [28][29][30].…”

Section: Introductionmentioning

confidence: 98%

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Rab3D regulates amylase levels, not agonist-induced amylase release, in AR42J cells

Limi

Ojakian

Raffaniello

2012

Cellular and Molecular Biology Letters

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Rab3D is a low molecular weight GTP-binding protein that associates with secretory granules in exocrine cells. AR42J cells are derived from rat pancreatic exocrine tumor cells and develop an acinar cell-like phenotype when treated with dexamethasone (Dex). In the present study, we examined the role of Rab3D in Dex-treated AR42J cells. Rab3D expression and localization were analyzed by subcellular fractionation and immunoblotting. The role of Rab3D was examined by overexpressing myc-labeled wild-type-Rab3D and a constitutively active form of Rab3D (Rab3D-Q81L) in AR42J cells. We found that Rab3D is predominantly membrane-associated in AR42J cells and co-localizes with zymogen granules (ZG). Following CCK-8-induced exocytosis, amylase-positive ZGs appeared to move towards the periphery of the cell and co-localization between Rab3D and amylase was less complete when compared to basal conditions. Overexpression of WT, but not mutant Rab3D, resulted in an increase in cellular amylase levels. Overexpression of mutant and WT Rab3D did not affect granule morphology, CCK-8-induced secretion, longterm (48 hr) basal amylase release or granule density. We conclude that Rab3D is not involved in agonist-induced exocytosis in AR42J cells. Instead, Rab3D may regulate amylase content in these cells.

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