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

Mai Sato, Yasunori Mori, Takahide Matsui, Ryo Aoki, Manami Oya, Yu Yanagihara, Mitsunori Fukuda, Takashi Tsuboi

Research output: Contribution to journalArticlepeer-review

18 Citations (Scopus)

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 Ca 2+-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.

Original languageEnglish
Pages (from-to)171-181
Number of pages11
JournalJournal of Neurochemistry
Volume114
Issue number1
DOIs
Publication statusPublished - 2010 Jul

Keywords

  • C2 domain
  • Dense-core vesicle
  • Doc2
  • Exocytosis
  • SNAP-25
  • Syntaxin-1a

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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