RRC ID 48869
Author Sato M, Mori Y, Matsui T, Aoki R, Oya M, Yanagihara Y, Fukuda M, Tsuboi T.
Title Role of the polybasic sequence in the Doc2alpha C2B domain in dense-core vesicle exocytosis in PC12 cells.
Journal J Neurochem
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, Doc2alpha, Doc2beta, and Doc2gamma, in humans and mice. Doc2alpha, the best-characterized, brain-specific isoform, exhibits Ca(2+)-dependent phospholipid-binding activity through its C2A domain, and the Ca(2+)-binding activity is thought to be important for the regulation of Ca(2+)-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 Doc2alpha (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 Doc2alpha(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.
Volume 114(1)
Pages 171-81
Published 2010-7-1
DOI 10.1111/j.1471-4159.2010.06739.x
PII JNC6739
PMID 20403080
MeSH Amino Acid Sequence Animals Calcium-Binding Proteins / genetics Calcium-Binding Proteins / physiology* Cell Membrane / metabolism Cytosol / metabolism Exocytosis* Mice Molecular Sequence Data Mutation Nerve Tissue Proteins / genetics Nerve Tissue Proteins / physiology* PC12 Cells Protein Binding Protein Multimerization Protein Structure, Tertiary Protein Transport Rats Secretory Vesicles / metabolism* Synaptosomal-Associated Protein 25 / genetics Syntaxin 1 / genetics
IF 4.066
Times Cited 17
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY NEUROSCIENCES
Resource
DNA material pEF-T7-mouse Doc2 alpha (RDB15100)