Ca2+-dependent interaction of the growth-associated protein GAP-43 with the synaptic core complex.
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.
Haruta
T
T
Department of Biochemistry, Fukuoka University School of Medicine, Nanakuma, Jonan-ku, Fukuoka 814-80, Japan.
Takami
N
N
Ohmura
M
M
Misumi
Y
Y
Ikehara
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D019922:GAP-43 Protein; D066298:In Vitro Techniques; D008561:Membrane Fusion / Q000502:physiology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology; D016716:PC12 Cells; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D051381:Rats; D013572:Synaptic Vesicles / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D050857:Synaptotagmins; D017830:Octoxynol; D011493:Protein Kinase C; D002118:Calcium