Role of the polybasic sequence in the Doc2alpha C2B domain in dense-core vesicle exocytosis in PC12 cells.
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.
Sato
Mai
M
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro, Tokyo, Japan.
Mori
Yasunori
Y
Matsui
Takahide
T
Aoki
Ryo
R
Oya
Manami
M
Yanagihara
Yu
Y
Fukuda
Mitsunori
M
Tsuboi
Takashi
T
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005089:Exocytosis; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D016716:PC12 Cells; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics; D050827:Syntaxin 1 / Q000235:genetics
D002135:Calcium-Binding Proteins; C490418:Doc2a protein, mouse; D009419:Nerve Tissue Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1