Analysis on novel regulatory proteins attached to the growth cone vesicles.

生长锥囊泡上附着的新型调节蛋白的分析。

• 批准号: 09680758
• 负责人: IGARASHI Michihiro
• 金额: $ 2.05万
• 依托单位: Gunma University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680758/
• 关键词: growth cone; SNARE complex; syntaxin; synapse; rab3A; rabphilin; exocytosis; シナプス終末; テトロトキシン; Rab3A サイクリング; GAP-43; 開口放出; シナプス前終末; C-キナーゼ; カルパイン

1) Growth cones are considered as the precursor of presynaptic terminals. To elucidate the minimal requirement of exocytotic molecular machinery, we examined the characteristics of exocytosis in growth cones using alpha-latrotoxin (alpha-LTX). The major biochemical difference between the adult presynptic terminal (SPS) and the isolated growth cone (IGC) was that rab3A was converted to GTP-form in the former, but remained GDP-form in the latter. We found that rabphilin was localized in much smaller amounts in IGC than in SPS.Following a supply of rabphilin, the IGC obtained as highly alpha-LTX-sensitivity as SPS, and the GDP-GTP conversion attached to rab3A on the GCV.In native IGCs, the GCVs had the SNAREs but riot NSF and alpha- SNAP ; while in the rabphilin-supplied IGC, the GCVs recruited NSF and alpha-SNAP, and the SNARE-NSF-SNAP complex was formed on the GCVs. Our results suggest that rab3A cycling develops later than the SNARE by accumulation of rabphilin, and that it is involved in determining the efficiency of exocytosis forced by alpha-LTX.2) Although growth cones responds to various modulators of neurite outgrowth, the signal-transducing mechanisms for these modulators in growth cones are unclear. I examined L-type VSCC-dependent signaling p-athways. In intact IGCs, Bay K 8644 (BK) induced much more rapid elevation of [Ca^<2+>] than that in SPS.Ca^<2+>-dependent phosphorylation of GAP-43 and MAROKS protein by protein kinase C (PKC) was enhanced in the JGC by BK, resulting in the release of these proteins from the membrane, which is consistent with our recent report. In addition, the Ca^<2+>-dependent degradation of brain spectrin by calpain was also enhanced by BK or GABA, consequently inducing the release of alpha-actinin from the membrane skeleton of growth cones. The activities of PKC and calpain were not inhibited by inhibitors of the other.

1)生长锥被认为是突触前终末的前身。为了阐明胞吐分子机制的最低要求，我们使用α-latrotoxin（α-LTX）研究了生长锥中胞吐的特征。成虫合成前终末（SPS）和离体生长锥（IGC）的主要生化差异是前者rab 3A转化为GTP形式，而后者仍为GDP形式。结果表明，IGC中rabphilin的表达量明显低于SPS，IGC对α-LTX的敏感性与SPS相当，GCV上的rab 3A也能促进GDP-GTP的转化。而在rabphilin供应的IGC中，GCV招募NSF和α-SNAP，并且在GCV上形成SNARE-NSF-SNAP复合物。我们的结果表明rab 3A循环通过rabphilin的积累比SNARE发展得晚，并且它参与决定由α-LTX强制的胞吐作用的效率。2）尽管生长锥响应于神经突生长的各种调节剂，但是生长锥中这些调节剂的信号转导机制尚不清楚。我检查了L型VSCC依赖性信号传导p途径。Bay K 8644（BK）可使完整IGCs的[Ca^2+]升高速度明显快于SPS，BK可增强JGC中蛋白激酶C（PKC）对GAP-43和MAROKS蛋白的Ca^2+依赖性磷酸化，从而使这些蛋白从膜上释放出来，这与我们最近的报道一致。此外，BK或GABA还可增强钙蛋白酶对脑血影蛋白的Ca^<2+>依赖性降解，从而诱导生长锥膜骨架释放α-辅肌动蛋白。PKC和钙蛋白酶的活性不受其他抑制剂的抑制。

项目成果

Ohbayasi K.et al.: "Shimulation of L-type Ca^<2+> channels in growd cored activated two independent signaling natuways." J.Neurosci.Res.51(6). 682-696 (1998)

Ohbayasi K.等人：“生长核心中L型Ca^2通道的刺激激活了两个独立的信号传导自然方式。”

Igarashi M: "From growth cone to synpase." Tanapkusitu-Kakusan-Koso(in Japanese). (in press). (1999)

五十岚 M：“从生长锥到突触。”

Igarashi M: "Molecular mechanisms of axonal growth." Igaku-no-Ayumi. 180(2). 138-140 (1997)

Igarashi M：“轴突生长的分子机制。”

五十嵐道弘: "軸索成長に関与する情報伝達" 生体の科学. 48(6). 542-554 (1997)

Michihiro Igarashi：“轴突生长中涉及的信息传输”生物科学 48（6）（1997）。

Igarashi M,Tagaya M,Komiya Y: "The soluble N-ethylmaleimide-sensitive factor attachment protein receptor(SNARE) complex in growth cones : molecular aspects of the axon terminal development." J.Neurosci.17(4). 1460-1470 (1997)

Igarashi M、Tagaya M、Komiya Y：“生长锥中的可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体 (SNARE) 复合物：轴突末端发育的分子方面。”