Hrs interacts with SNAP-25 and regulates Ca(2+)-dependent exocytosis.
Synaptosome-associated protein of 25 kDa (SNAP-25) is a neuronal membrane protein essential for synaptic vesicle exocytosis. To investigate the mechanisms by which SNAP-25 mediates neurosecretion, we performed a search for proteins that interact with SNAP-25 using a yeast two-hybrid screen. Here, we report the isolation and characterization of a SNAP-25-interacting protein that is the rat homologue of mouse hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Hrs specifically interacts with SNAP-25, but not SNAP-23/syndet. The association of Hrs and SNAP-25 is mediated via coiled-coil interactions. Using an Hrs-specific antibody, we have shown that Hrs is highly enriched in brain, where it codistributes with SNAP-25 in most brain regions. Subcellular fractionation studies demonstrate that in brain, Hrs exists in both cytosolic and membrane-associated pools. Studies using indirect immunofluorescence and confocal microscopy reveal that, in addition to early endosomes, Hrs is also localized to large dense-core secretory granules and synaptic-like microvesicles in nerve growth factor-differentiated PC12 cells. Moreover, overexpression of Hrs in PC12 cells inhibits Ca(2+)-dependent exocytosis. These results suggest that Hrs is involved in regulation of neurosecretion through interaction with SNAP-25.
Kwong
J
J
Department of Pharmacology, Bowles Center for Alcohol Studies, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA.
Roundabush
F L
FL
Hutton Moore
P
P
Montague
M
M
Oldham
W
W
Li
Y
Y
Chin
L S
LS
Li
L
L
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000502:physiology; D056827:Endosomal Sorting Complexes Required for Transport; D005089:Exocytosis / Q000502:physiology*; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000502:physiology*; D016716:PC12 Cells; D010750:Phosphoproteins / Q000502:physiology*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D050825:Synaptosomal-Associated Protein 25
D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; C494209:Snap25 protein, mouse; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; D002118:Calcium