Hrs interacts with SNAP-25 and regulates Ca2+-dependent exocytosis

Synaptosome-associated protein of 25 kDa (SNAP-25) is a neuronal membrane p rotein essential for synaptic vesicle exocytosis. To investigate the mechan isms by which SNAP-25 mediates neurosecretion, we performed a search for pr oteins 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 tyro sine kinase substrate (Hrs). Hrs specifically interacts with SNAP-25, but n ot SNAP-23/syndet. The. association of Hrs and SNAP-25 is mediated via coil ed-coil interactions. Using an I-Irs-specific antibody, we have shown that Hrs is highly enriched in brain, where it codistributes with SNAP-25 in mos t brain regions. Subcellular fractionation studies demonstrate that in brai n, Hrs exists in both cytosolic and membrane-associated pools, Studies usin g indirect immunofluorescence and confocal microscopy reveal that, in addit ion to early endosomes, Hrs is also localized to large dense-core secretory granules and synaptic-like microvesicles in nerve growth factor-differenti ated PC12 cells, Moreover, overexpression of Hrs in PC12 cells inhibits Ca2 +-dependent exocytosis, These results suggest that Hrs is involved in regul ation of neurosecretion through interaction,vith SNAP-25.