Ca2+-dependent interaction of the growth-associated protein GAP-43 with the synaptic core complex

• Published in: Biochemical journal Vol. 325 ( Pt 2); no. 2; pp. 455 - 463
• Main Authors: Haruta, T, Takami, N, Ohmura, M, Misumi, Y, Ikehara, Y
• Format: Journal Article
• Language: English
• Published: England 15-07-1997
• Subjects: Amino Acid Sequence; Animals; Brain - metabolism; Calcium - pharmacology; Calcium-Binding Proteins; Calmodulin - metabolism; Cell Line; GAP-43 Protein; In Vitro Techniques; Membrane Fusion - physiology; Membrane Glycoproteins - metabolism; Membrane Proteins - metabolism; Molecular Sequence Data; Nerve Growth Factors - pharmacology; Nerve Tissue Proteins - metabolism; Octoxynol - pharmacology; PC12 Cells; Phosphorylation; Protein Kinase C - metabolism; Qa-SNARE Proteins; R-SNARE Proteins; Rats; Synaptic Vesicles - metabolism; Synaptosomal-Associated Protein 25; Synaptotagmins
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3250455

The synaptic vesicle exocytosis occurs by a highly regulated mechanism: syntaxin and 25 kDa synaptosome-associated protein (SNAP-25) are assembled with vesicle-associated membrane protein (VAMP) to form a synaptic core complex and then synaptotagmin participates as a Ca2+ sensor in the final step of membrane fusion. The 43 kDa growth-associated protein GAP-43 is a nerve-specific protein that is predominantly localized in the axonal growth cones and presynaptic terminal membrane. In the present study we have examined a possible interaction of GAP-43 with components involved in the exocytosis. GAP-43 was found to interact with syntaxin, SNAP-25 and VAMP in rat brain tissues and nerve growth factor-dependently differentiated PC12 cells, but not in undifferentiated PC12 cells. GAP-43 also interacted with synaptotagmin and calmodulin. These interactions of GAP-43 could be detected only when chemical cross-linking of proteins was performed before they were solubilized from the membranes with detergents, in contrast with the interaction of the synaptic core complex, which was detected without cross-linking. Experiments in vitro showed that the interaction of GAP-43 with these proteins occurred Ca2+-dependently; its maximum binding with the core complex was observed at 100 microM Ca2+, whereas that of syntaxin with synaptotagmin was at 200 microM Ca2+. These values of Ca2+ concentration are close to that required for the Ca2+-dependent release of neurotransmitters. Furthermore we observed that the interaction in vitro of GAP-43 with the synaptic core complex was coupled with protein kinase C-mediated phosphorylation of GAP-43. Taken together, our results suggest a novel function of GAP-43 that is involved in the Ca2+-dependent fusion of synaptic vesicles.