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J. Biol. Chem., Vol. 282, Issue 31, 22887-22899, August 3, 2007

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Receptor-mediated Regulation of Tomosyn-Syntaxin 1A Interactions in Bovine Adrenal Chromaffin Cells^*

Svetlana E. Gladycheva¹, Alice D. Lam¹², Jiang Liu, Matthew D'Andrea-Merrins, Ofer Yizhar, Stephen I. Lentz^¶, Uri Ashery, Stephen A. Ernst^||, and Edward L. Stuenkel³

From the Department of Molecular and Integrative Physiology, ^||Department of Cell and Developmental Biology, and ^¶Michigan Diabetes Research and Training Center, University of Michigan, Ann Arbor, Michigan 48109 and the Department of Neurobiochemistry, Tel Aviv University, Tel Aviv, 69978 Israel

Tomosyn, a soluble R-SNARE protein identified as a binding partner of the Q-SNARE syntaxin 1A, is thought to be critical in setting the level of fusion-competent SNARE complexes for neurosecretion. To date, there has been no direct evaluation of the dynamics in which tomosyn transits through tomosyn-SNARE complexes or of the extent to which tomosyn-SNARE complexes are regulated by secretory demand. Here, we employed biochemical and optical approaches to characterize the dynamic properties of tomosyn-syntaxin 1A complexes in live adrenal chromaffin cells. We demonstrate that secretagogue stimulation results in the rapid translocation of tomosyn from the cytosol to plasma membrane regions and that this translocation is associated with an increase in the tomosyn-syntaxin 1A interaction, including increased cycling of tomosyn into tomosyn-SNARE complexes. The secretagogue-induced interaction was strongly reduced by pharmacological inhibition of the Rho-associated coiled-coil forming kinase, a result consistent with findings demonstrating secretagogue-induced activation of RhoA. Stimulation of chromaffin cells with lysophosphatidic acid, a nonsecretory stimulus that strongly activates RhoA, resulted in effects on tomosyn similar to that of application of the secretagogue. In PC-12 cells overexpressing tomosyn, secretagogue stimulation in the presence of lysophosphatidic acid resulted in reduced evoked secretory responses, an effect that was eliminated upon inhibition of Rho-associated coiled-coil forming kinase. Moreover, this effect required an intact interaction between tomosyn and syntaxin 1A. Thus, modulation of the tomosyn-syntaxin 1A interaction in response to secretagogue activation is an important mechanism allowing for dynamic regulation of the secretory response.

Received for publication, February 28, 2007 , and in revised form, May 31, 2007.

^* This work was supported by National Institutes of Health (NIH) Grants NS39914 and NS053978 (to E. L. S.), NIDDK, NIH, Grants P30DK 34933 and P60DK20572 (to S. A. E.), and National Research Service Award RSA NS053263 (to A. D. L.). This work utilized the Morphology and Image Analysis Core of the Michigan Diabetes Research and Training Center funded by NIDDK, National Institutes of Health Grant NIH5P60 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.

¹ These two authors contributed equally to this work.

² Supported in part by National Institutes of Health Medical Scientist Training Grant GM007863.

³ To whom correspondence should be addressed: 7807 Medical Sciences II Bldg., Dept. of Molecular and Integrative Physiology, The Medical School, University of Michigan, Ann Arbor, MI 48109-0622. Tel.: 734-763-4477; Fax: 734-936-8813; E-mail: esterm{at}umich.edu.

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				A. D. Lam, P. Tryoen-Toth, B. Tsai, N. Vitale, and E. L. Stuenkel SNARE-catalyzed Fusion Events Are Regulated by Syntaxin1A-Lipid Interactions Mol. Biol. Cell, February 1, 2008; 19(2): 485 - 497. [Abstract] [Full Text] [PDF]