HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 50, 50053-50060, December 12, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/50/50053    most recent M309910200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chen, X. Articles by Williams, J. A. Search for Related Content PubMed PubMed Citation Articles by Chen, X. Articles by Williams, J. A. Social Bookmarking                      What's this?

Dominant Negative Rab3D Mutants Reduce GTP-bound Endogenous Rab3D in Pancreatic Acini^*

Xuequn Chen, Stephen A. Ernst¶, and John A. Williams||

From the Departments of Molecular and Integrative Physiology, ^¶Cell and Developmental Biology, and ^||Internal Medicine, The University of Michigan, Ann Arbor, Michigan 48109

Two dominant negative mutants of Rab3D, N135I and T36N were recently reported to inhibit the early phase of regulated amylase secretion from mouse pancreatic acini (Chen, X., Edwards, J. A., Logsdon, C. D., Ernst, S. A., and Williams, J. A. (2002) J. Biol. Chem. 277, 18002–18009). Immunocytochemical studies showed that while the wild-type Rab3D localized to zymogen granules, the two dominant negative mutants did not localize to granules and were primarily in the basolateral regions of the cell. The present study, therefore, evaluated 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. 78.9 ± 4.5% of total endogenous Rab3D was found in the GTP-bound form. Overexpression of HA-tagged Rab3D, and its Q81L, N135I, and T36N mutants had no effect on the total amount of endogenous Rab3D. However, the dominant negative mutants, T36N and N135I, reduced GTP-bound endogenous Rab3D by 70.0 ± 3.5% and 72.7 ± 1.2%, respectively, while the wild-type Rab3D and Q81L mutant 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. The dominant negative Rab3D did not affect the amount of endogenous Rab3D on purified zymogen granules as assessed by either Western blotting or immunocytochemistry, but reduced the GTP-bound form by 78.6 ± 3.3%. 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.

Received for publication, September 5, 2003 , and in revised form, September 26, 2003.

^* This work was supported by National Institutes of Health Grant DK41122 (to J. A. W.), the Michigan Gastrointestinal Peptide Center, P30 DK34933, and the Michigan Diabetes Research and Training Center, P60 DK20572. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence may be addressed: Dept. of Molecular and Integrative Physiology, The University of Michigan, 7734 Medical Sciences Bldg. II, Ann Arbor, MI 48109-0622. Tel.: 734-764-9456; Fax: 734-936-8813; E-mail: xuequnc{at}umich.edu or jawillms{at}umich.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. E. Sabbatini, X. Chen, S. A. Ernst, and J. A. Williams Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion J. Biol. Chem., August 29, 2008; 283(35): 23884 - 23894. [Abstract] [Full Text] [PDF]

				E. Evans, W. Zhang, G. Jerdeva, C.-Y. Chen, X. Chen, S. F. Hamm-Alvarez, and C. T. Okamoto Direct interaction between Rab3D and the polymeric immunoglobulin receptor and trafficking through regulated secretory vesicles in lacrimal gland acinar cells Am J Physiol Cell Physiol, March 1, 2008; 294(3): C662 - C674. [Abstract] [Full Text] [PDF]

				G. T. Gurda, L. Guo, S.-H. Lee, J. D. Molkentin, and J. A. Williams Cholecystokinin Activates Pancreatic Calcineurin-NFAT Signaling In Vitro and In Vivo Mol. Biol. Cell, January 1, 2008; 19(1): 198 - 206. [Abstract] [Full Text] [PDF]

				J. Zhang, K. L. Schulze, P. R. Hiesinger, K. Suyama, S. Wang, M. Fish, M. Acar, R. A. Hoskins, H. J. Bellen, and M. P. Scott Thirty-One Flavors of Drosophila Rab Proteins Genetics, June 1, 2007; 176(2): 1307 - 1322. [Abstract] [Full Text] [PDF]

				H. Kondo, R. Shirakawa, T. Higashi, M. Kawato, M. Fukuda, T. Kita, and H. Horiuchi Constitutive GDP/GTP Exchange and Secretion-dependent GTP Hydrolysis Activity for Rab27 in Platelets J. Biol. Chem., September 29, 2006; 281(39): 28657 - 28665. [Abstract] [Full Text] [PDF]

				C. M. Babbey, N. Ahktar, E. Wang, C. C.-H. Chen, B. D. Grant, and K. W. Dunn Rab10 Regulates Membrane Transport through Early Endosomes of Polarized Madin-Darby Canine Kidney Cells Mol. Biol. Cell, July 1, 2006; 17(7): 3156 - 3175. [Abstract] [Full Text] [PDF]

				X. Chen, A. K. Walker, J. R. Strahler, E. S. Simon, S. L. Tomanicek-Volk, B. B. Nelson, M. C. Hurley, S. A. Ernst, J. A. Williams, and P. C. Andrews Organellar Proteomics: Analysis of Pancreatic Zymogen Granule Membranes Mol. Cell. Proteomics, February 1, 2006; 5(2): 306 - 312. [Abstract] [Full Text] [PDF]

				R. C. De Lisle, O. Norkina, E. Roach, and D. Ziemer Expression of pro-Muclin in pancreatic AR42J cells induces functional regulated secretory granules Am J Physiol Cell Physiol, November 1, 2005; 289(5): C1169 - C1178. [Abstract] [Full Text] [PDF]

				N. J. Pavlos, J. Xu, D. Riedel, J. S. G. Yeoh, S. L. Teitelbaum, J. M. Papadimitriou, R. Jahn, F. P. Ross, and M. H. Zheng Rab3D Regulates a Novel Vesicular Trafficking Pathway That Is Required for Osteoclastic Bone Resorption Mol. Cell. Biol., June 15, 2005; 25(12): 5253 - 5269. [Abstract] [Full Text] [PDF]