TY - JOUR
T1 - Role of the polybasic sequence in the Doc2α C2B domain in dense-core vesicle exocytosis in PC12 cells
AU - Sato, Mai
AU - Mori, Yasunori
AU - Matsui, Takahide
AU - Aoki, Ryo
AU - Oya, Manami
AU - Yanagihara, Yu
AU - Fukuda, Mitsunori
AU - Tsuboi, Takashi
N1 - Copyright:
Copyright 2010 Elsevier B.V., All rights reserved.
PY - 2010/7
Y1 - 2010/7
N2 - 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.
AB - 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.
KW - C2 domain
KW - Dense-core vesicle
KW - Doc2
KW - Exocytosis
KW - SNAP-25
KW - Syntaxin-1a
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U2 - 10.1111/j.1471-4159.2010.06739.x
DO - 10.1111/j.1471-4159.2010.06739.x
M3 - Article
C2 - 20403080
AN - SCOPUS:77953312434
VL - 114
SP - 171
EP - 181
JO - Journal of Neurochemistry
JF - Journal of Neurochemistry
SN - 0022-3042
IS - 1
ER -