![]() In this study we investigated the SNAP-25-binding site in the C2B domain of rabphilin and produced two types of rabphilin mutants, a KQ mutant that lacks Ca 2+-independent SNAP-25-binding and a D34N mutant that lacks putative Ca 2+-binding sites ( Ubach et al. 1999), the C2B ligand that is critical for the vesicle docking step remains to be identified. However, as the C2B domain of rabphilin possesses Ca 2+-dependent and -independent binding properties, and is likely to possess distinct ligand binding sites ( Yamaguchi et al. We previously suggested that a direct interaction between the rabphilin C2B domain and a plasma membrane protein SNAP-25 (synaptosome-associated protein of 25 kDa) is involved in the dense-core vesicle docking step. C2B domain) has been found to be crucial for docking activity, because a mutant rabphilin lacking the C2B domain does not mediate vesicle docking to the plasma membrane ( Tsuboi and Fukuda 2005). By contrast, rabphilin has been shown to increase ‘releasable’ docked vesicles, and the second C2 domain (i.e. 2005 Tsuboi and Fukuda 2006b), and most of them do not undergo exocytosis even under stimulating conditions ( Coppola et al. Slp4-a has been shown to increase the number of ‘inert’ vesicles docked to the plasma membrane, through interaction with Munc18-1 2004 Fukuda 2005), they have opposite effects on hormone secretion. Although Slp4-a and rabphilin basically share the same domain structures, an N-terminal Rab-binding domain and C-terminal tandem C2 domains ( Cheviet et al. 2004), promote dense-core vesicle docking to the plasma membrane ( Tsuboi and Fukuda 2005, 2006b). We have recently shown that Rab3A and Rab27A, but not 37 other Rabs, cooperatively regulate the docking step of dense-core vesicle exocytosis in neuroendocrine PC12 cells ( Tsuboi and Fukuda 2006a), and that expression of two effector molecules, synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a and rabphilin, both of which bind Rab3A and Rab27A in vitro ( Kuroda et al. 1996 Geppert and Südhof 1998 Cheviet et al. Two small GTPases, Rab3 and Rab27, are abundantly expressed on dense-core vesicles and have been shown to control their exocytosis through specific interaction with effector molecules (reviewed in Takai et al. Hormone secretion is achieved by recruitment, docking and fusion of dense-core vesicles with the plasma membrane, and a variety of molecules, including soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and small GTPase Rab, are thought to control dense-core vesicle exocytosis (reviewed in Burgoyne and Morgan 2003). Peptide hormones are stored in large dense-core vesicles in neuroendocrine cells, and are secreted by exocytosis in response to extracellular stimuli. pH-insensitive yellow fluorescent protein.soluble N-ethylmaleimide-sensitive factor attachment protein receptor.synaptosome-associated protein of 25 kDa.sodium dodecyl sulfate–polyacrylamide gel electrophoresis.These results indicate that the polybasic sequence in the C2B domain functions as an effector domain for SNAP-25 and controls the number of ‘releasable’ vesicles docked to the plasma membrane. A rabphilin(KQ) mutant that completely lacks SNAP-25-binding activity significantly decreased the number of plasma-membrane-docked vesicles and strongly inhibited high-KCl-induced dense-core vesicle exocytosis. We also investigated the effect of Lys→Gln (KQ) mutations in the polybasic sequence of the C2B domain on vesicle dynamics by total internal reflection fluorescence microscopy in individual PC12 cells. ![]() In this study we demonstrated by a mutation analysis that the polybasic sequence (587 KKAKHKTQIKKK 598) in the C2B domain of rabphilin is required for SNAP-25 binding, and that the Asp residues in the Ca 2+-binding loop 3 (D628 and D630) of the C2B domain are not required. However, the physiological significance of the rabphilin–SNAP-25 interaction in the vesicle-docking step has never been elucidated. Rabphilin is generally thought to be involved in the regulation of secretory vesicle exocytosis in neurons and neuroendocrine cells, and it has recently been hypothesized that the C2B domain of rabphilin promotes the docking of dense-core vesicles to the plasma membrane through simultaneous interaction with a vesicle protein, Rab3A/27A, and a plasma membrane protein, SNAP-25 (synaptosome-associated protein of 25 kDa). ![]()
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