Identification of a cellubrevin/vesicle associated membrane protein 3 homologue in human platelets

Blood. 1999 Jan 15;93(2):571-9.

Abstract

Several studies suggest membrane trafficking events are mediated by integral, membrane proteins from both transport-vesicle and target membranes, called v- and t-SNAREs (SNAp REceptors), respectively. Previous experiments using antibodies to synaptobrevin/vesicle associated membrane protein (VAMP) 1, 2, or rat cellubrevin failed to detect these v-SNAREs in human platelets, although membrane proteins from these cells could support 20S complex formation. To identify v-SNAREs in platelets, we used a polymerase chain reaction (PCR) approach with degenerate primers to amplify potential VAMP-like v-SNAREs. A cDNA encoding a novel v-SNARE was isolated from a human megakaryocyte cDNA library. Termed human cellubrevin (Hceb), this protein has greater than 93% identity with human VAMP 1, 2, and rat cellubrevin over the conserved core region, but has a unique N-terminal domain. Northern blot analysis showed that the 2. 5-kB mRNA encoding Hceb is expressed in every human tissue tested. Hceb from detergent-solubilized platelet membranes, participated in alpha-SNAP-dependent 20S complex formation and adenosine triphosphate (ATP)-dependent disassembly, showing that Hceb can act as a v-SNARE in platelets. Immunofluorescence microscopy, using an anti-Hceb antibody showed a punctate, intracellular staining pattern in platelets, megakaryocytes, and HEK-293 cells. This same pattern was observed in surface-activated platelets even though all dense core and most alpha-granule contents had been released. These data suggest that Hceb may reside on a platelet organelle that is not primarily involved in the exocytic pathway.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Blood Platelets / chemistry*
  • Blotting, Northern
  • DNA, Complementary / analysis
  • DNA, Complementary / chemistry
  • Fluorescent Antibody Technique
  • Humans
  • Megakaryocytes / chemistry
  • Membrane Proteins / analysis*
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • RNA, Messenger / analysis
  • Rats
  • Sequence Homology
  • Tissue Distribution
  • Vesicle-Associated Membrane Protein 3

Substances

  • DNA, Complementary
  • Membrane Proteins
  • RNA, Messenger
  • Vesicle-Associated Membrane Protein 3

Associated data

  • GENBANK/U64520