VAMP Family with special emphasis on VAMP8/Endobrevin

Vesicle Associated Membrane Proteins (VAMPs) are classified as R SNAREs since they contribute an Arginine residue to the 0^th layer of the core complex. Till date 7 mammalian VAMPS have been identified and are named VAMP 1-5, 7 and 8. VAMP 1-3 and 5 are toxin sensitive VAMPs while VAMP4, VAMP7 and VAMP8 (Endobrevin) are insensitive to tetanus toxin treatment.

VAMPs 1-5 and 7: The first member of the R-SNARE VAMP family to be known was a 120 amino acid long membrane protein, isolated from the synaptic vesicles of Torpedo californica (Trimble et al. 1988). Using cDNA clone of this VAMP, two related genes named VAMP1 and VAMP2 were identified in rat brain (Elferink et al. 1989). Eventually, VAMP1 was identified in bovine brain and was found completely identical to VAMP2 of the rat brain (Sudhof et al. 1989). VAMP1 and VAMP2 are called synaptobrevin 1 and 2

Chapter 1. Introduction

respectively and are highly expressed in neuron and endocrine cells. Synaptobrevins are involved in exocytosis of synaptic vesicles (Jahn and Sudhof 1994).

A non neuronal homologue of synaptobrevins was identified and named VAMP3/Cellubrevin. It was found ubiquitously expressed in all the cells and tissues (hence the name cellubrevin) (Mcmahon et al. 1993a). Several independent studies have been done to assign a physiological role to VAMP3. It has been localized on the membranes of pancreatic secretory vesicles and is implicated in regulating the Ca²⁺ mediated secretion of insulin (Regazzi et al. 1995). VAMP3 was also been localized to platelets and implicated in release of platelet granules (Polgar et al. 2002). In line with these reports, recently VAMP3 has been shown to possibly carry out exocytosis of platelet granules in the absence of the primary platelet v-SNARE VAMP8 (Ren et al. 2006).

VAMP4 was identified in EST database search and has a broad tissue expression profile. It was localized to the Golgi-TGN in the NRK cells (Advani et al. 1998) and tubular and vesicular membranes of the TGN in PC12 cells (Steegmaier et al. 1999a). VAMP4 was shown to function in the SNARE complex that mediates retrograde trafficking from early endosomes to TGN (Ref section 1.2.5) (Kreykenbohm et al. 2002).

VAMP5 was identified in EST database search. It was found to be increased during in vitro myogenesis in C2C12 cells and was preferentially expressed in muscles and heart.

VAMP5 was found associated with plasma membrane and intracellular vesicular structures in myotubes (Zeng et al. 1998a).

VAMP7 also called toxin insensitive VAMP (TI-VAMP) was identified in EST database search (Advani et al. 1998). It was found to be abundant on the membranes of the trans Golgi network and the late endosomes (Advani et al. 1999) and mediates vesicular transport from the late endosome to the lysosomes (Ref section 1.2.5) (Advani et al. 1999; Ward et al. 2000). VAMP7 was shown to play a critical role in the onset of phagocytosis in macrophages (Braun et al. 2004) and vesicular transport in neurite outgrowth (Martinez-Arca et al. 2000).

VAMP8: VAMP8 also named endobrevin was first identified in EST database search (Wong et al. 1998). Vamp8 gene in mice comprises of four exons. The first exon is non-coding. The second codon contains the 5’UTR and the start codon that encodes the first

Chapter 1. Introduction

amino acid M1 (Methionine). Exon 3 encodes amino acid E2 (Glutamic acid) - T54 (Threonine) and exon 4 encodes S55 (Serine)-T101 (Threonine) and the 3’UTR. The SNARE motif of VAMP8 is encoded in part by exon 3 (aa 13-54) and exon 4 (aa 55-62).

The cytoplasmic domain is predicted to be encoded by residues 1-75 (Wong et al. 1998).

The mouse Vamp8 gene was found positioned on chromosome 2 while in humans on chromosome 2 (Sikorra et al. 2006).

VAMP8 is a 15kDa, integral membrane protein (Wong et al. 1998) The amino acid sequence of VAMP8 was found to be 32% identical to synaptobrevin/VAMP1, 33%

identical to synaptobrevin/VAMP-2 and had 31% identity to VAMP3/cellubrevin. It does not contain the conserved toxin cleavage site and is therefore insensitive to tetanus toxin and botulinum toxin (Wong et al. 1998). By immunogold labeling VAMP8 was found enriched on early endosomes (Wong et al. 1998). Sub cellular fractionation revealed showed VAMP8 to be present in same fractions as asialoglycoprotein receptor corresponding to the early endosomes (hence the name endobrevin) (Wong et al. 1998). In line with these reports, using immunofluorescence staining, VAMP8 was localized abundantly at the membranes of early and late endosomes (Antonin et al. 2000b; Wong et al. 1998). Using two independent approaches, anti-VAMP8 antibody and recombinant VAMP8 protein for inhibiting SNARE assembly, VAMP8 was shown to mediate homotypic fusion of early and late endosomes in PC12 cells (Antonin et al. 2000c). Later, using content mixing assay it was shown that antibody against VAMP8 inhibits only the late endosomal fusion and not the early endosomal fusion. The same result was confirmed by microinjecting Fab fragments of VAMP8 as well as its complex partners syntaxin 8, syntaxin7 and Vti1b. The Fab fragments of each of VAMP8 and other three complex partners resulted in a profound delay in the trafficking of EGF to the lysosomes thereby confirming that VAMP8 is involved in late endosomal fusion (Antonin et al. 2000b).

Besides the late endosomal trafficking, several studies have shown VAMP8 to mediate regulated exocytosis events in several cell types. Using pancreatic acinar cells from VAMP8^-/^- mice, VAMP8 was shown to be important for regulated exocytosis of zymogen granules in a complex with SNAP23 and synatxin4 (Wang et al. 2004). VAMP8 was found on the human platelets and was suggested to mediate regulated secretion of platelet

Chapter 1. Introduction

granules (Polgar et al. 2002) and mast cells (Paumet et al. 2000b). In line with these suggestions, using platelets derived from VAMP8^-/^- mice it was shown that VAMP8 is the the primary R-SNAREs involved in the release from dense core granules, alpha granules, and lysosomes from the platelets (Ren et al. 2006). VAMP8 was also shown to be critical for the terminal step of cytokinesis in normal rat kidney (NRK) cells (Low et al. 2003).