title: Receptor-mediated regulation of tomosyn-syntaxin 1A interactions in bovine adrenal chromaffin cells.
abstract: 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.
authors.affiliation: Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Svetlana E
authors.initials: SE
authors.lastname: Gladycheva
authors.forename: Alice D
authors.initials: AD
authors.lastname: Lam
authors.forename: Jiang
authors.initials: J
authors.lastname: Liu
authors.forename: Matthew
authors.initials: M
authors.lastname: D'Andrea-Merrins
authors.forename: Ofer
authors.initials: O
authors.lastname: Yizhar
authors.forename: Stephen I
authors.initials: SI
authors.lastname: Lentz
authors.forename: Uri
authors.initials: U
authors.lastname: Ashery
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Ernst
authors.forename: Edward L
authors.initials: EL
authors.lastname: Stuenkel
mesh_terms: D000311:Adrenal Glands / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019439:Chromaffin Cells / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D051938:Munc18 Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D016716:PC12 Cells; D021381:Protein Transport; D050683:R-SNARE Proteins / Q000737:chemistry*; D051381:Rats; D050827:Syntaxin 1 / Q000737:chemistry* / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; C484767:STXBP5 protein, human; D050827:Syntaxin 1; D020741:rho GTP-Binding Proteins