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Free Content Role of the polybasic sequence in the Doc2α C2B domain in dense-core vesicle exocytosis in PC12 cells

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J. Neurochem. (2010) 114, 171–181. Abstract

The double C2 (Doc2) family is characterized by an N-terminal Munc13-1-interacting domain and C-terminal tandem C2 domains, and it comprises three isoforms, Doc2α, Doc2β, and Doc2γ, in humans and mice. Doc2α, the best-characterized, brain-specific isoform, exhibits Ca2+-dependent phospholipid-binding activity through its C2A domain, and the Ca2+-binding activity is thought to be important for the regulation of Ca2+-dependent exocytosis. In contrast to the C2A domain, however, nothing is known about the physiological functions of the C2B domain in regulated exocytosis. In this study, we demonstrated by a mutation analysis that the polybasic sequence in the C2B domain of Doc2α (306 KKSKHKTCVKKK 317) is required for binding of syntaxin-1a/synaptosome-associated protein of 25 kDa (SNAP-25) heterodimer. We also investigated the effect of Lys-to-Gln (named KQ) mutations in the polybasic sequence of the C2B domain on vesicle dynamics by total internal reflection fluorescence microscopy in PC12 cells. A Doc2α(KQ) mutant, which lacks binding activity toward syntaxin-1a/SNAP-25 heterodimer, significantly decreased the number of plasma membrane-docked vesicles before stimulation and strongly inhibited high-KCl-induced exocytosis from the plasma membrane-docked vesicles. These results indicate that the polybasic sequence in the C2B domain functions as a binding site for syntaxin-1a/SNAP-25 heterodimer and controls the number of ‘readily releasable’ vesicles in neuroendocrine cells.
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Keywords: C2 domain; Doc2; SNAP-25; dense-core vesicle; exocytosis; syntaxin-1a

Document Type: Research Article

Affiliations: 1: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro, Tokyo, Japan 2: Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi, Japan

Publication date: 01 July 2010

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