The C2B Domain of Rabphilin Directly Interacts with SNAP-25 and Regulates the Docking Step of Dense Core Vesicle Exocytosis in PC12 Cells

Takashi Tsuboi,Mitsunori Fukuda

doi:10.1074/jbc.m507173200

Takashi Tsuboi, Mitsunori Fukuda

Open Access

https://doi.org/10.1074/jbc.m507173200

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Abstract

Rabphilin is a membrane trafficking protein on secretory vesicles that consists of an N-terminal Rab-binding domain and C-terminal tandem C2 domains. The N-terminal part of rabphilin has recently been shown to function as an effector domain for both Rab27A and Rab3A in PC12 cells (Fukuda, M., Kanno, E., and Yamamoto, A. (2004) J. Biol. Chem. 279, 13065-13075), but the function of the C2 domains of rabphilin during secretory vesicle exocytosis is largely unknown. In this study we investigated the interaction between rabphilin and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors, VAMP-2/synaptobrevin-2, syntaxin IA, and SNAP-25) and SNARE-associated proteins (Munc18-1 and Munc13-1) and found that the C2B domain of rabphilin, but not of other Rab27A-binding proteins with tandem C2 domains (i.e. Slp1-5), directly interacts with a plasma membrane protein, SNAP-25. The interaction between rabphilin and SNAP-25 occurs even in the absence of Ca(2+) (EC(50) = 0.817 microm SNAP-25), but 0.5 mm Ca(2+) increases the affinity for SNAP-25 2-fold (EC(50) = 0.405 microm SNAP-25) without changing the B(max) value (1.06 mol of SNAP-25/mol of rabphilin). Furthermore, vesicle dynamics were imaged by total internal reflection fluorescence microscopy in a single PC12 cell expressing a lumen-targeted pH-insensitive yellow fluorescent protein (Venus), neuropeptide Y-Venus. Expression of the wild-type rabphilin in PC12 cells significantly increased the number of docked vesicles to the plasma membrane without altering the kinetics of individual secretory events, whereas expression of the mutant rabphilin lacking the C2B domain, rabphilin-DeltaC2B, decreased the number of docked vesicle or fusing at the plasma membrane. These findings suggest that rabphilin is involved in the docking step of regulated exocytosis in PC12 cells, possibly through interaction between the C2B domain and SNAP-25.

Highlights

Rab3 knock-out animals display distinct phenotypes in terms of neurotransmitter release [7, 8]
Co-purification of SNAP-25 and Rab27A with Rabphilin from PC12 Cell Lysates—To investigate the possible interaction between rabphilin and SNAREs, i.e. syntaxin I, SNAP-25, and VAMP-2, or SNARE-associated proteins, i.e. Munc13 and Munc18, GST-tagged rabphilin was transiently expressed in PC12 cells, and GST-rabphilin and rabphilininteracting molecules were affinity-purified on glutathione-Sepharose beads
The neuropeptide Y (NPY)-Venus-containing spots grew brighter and expanded suddenly during the release of the fluorescent peptide in response to high KCl stimulation [31], with an identical time course, in both the control cells and monomeric red fluorescent protein (mRFP)-rabphilin-expressing cells (Fig. 6B). These results strongly indicate that rabphilin promotes docking of dense core vesicles to the plasma membrane in PC12 cells rather than modulates vesicle fusion

Summary

Introduction

Rab3 knock-out animals display distinct phenotypes in terms of neurotransmitter release [7, 8]. Agarose beads coupled with T7-rabphilin were incubated with COS-7 cell lysates containing FLAGsyntaxin IA, FLAG-SNAP-25, FLAG-VAMP-2, or FLAG-Munc18-1, in the presence and absence of HA-Rab27A, and FLAG-tagged proteins and HA-Rab27A that had bound to the beads were detected by immunoblotting with the specific antibodies indicated (Fig. 2).

Results

Conclusion