Amphiphysin I Is Associated with Coated Endocytic Intermediates and Undergoes Stimulation-dependent Dephosphorylation in Nerve Terminals

作者：Rudolf Bauerfeind, Kohji Takei, Pietro V. De Camilli · 发表于：Journal of Biological Chemistry · 年份：1997 · DOI：10.1074/jbc.272.49.30984 · 被引用次数：164 · 研究领域：Cellular transport and secretion、Lipid Membrane Structure and Behavior、Neuroscience and Neuropharmacology Research

Amphiphysin I is an abundant presynaptic protein that interacts via its COOH-terminal src homology 3 (SH3) domain with the GTPase dynamin I and the inositol-5-phosphatase synaptojanin. Both dynamin I and synaptojanin I have a putative role in synaptic vesicle recycling and undergo rapid dephosphorylation in rat brain synaptosomes stimulated to secrete by a depolarizing stimulus. We show here that amphiphysin I also undergoes constitutive phosphorylation and stimulationdependent dephosphorylation. Dephosphorylation of amphiphysin I requires extracellular Ca2+ and is unaffected by pretreatment of synaptosomes with tetanus toxin. Thus, Ca2+ influx, but not synaptic vesicle exocytosis, is required for dephosphorylation. Dephosphorylation of amphiphysin I, like dephosphorylation of dynamin I and synaptojanin I, is inhibited by cyclosporin A and FK-506 (0.5 microM), two drugs that specifically block the Ca2+/calmodulin-dependent phosphatase 2B calcineurin, but not by okadaic acid (1 microM), which blocks protein phosphatases 1 and 2B. We also show by immunogold electron microscopy immunocytochemistry that amphiphysin I is localized in the nerve terminal cytomatrix and is partially associated with endocytic intermediates. These include the clathrin-coated buds and dynamin-coated tubules, which accumulate in nerve terminal membranes incubated in the presence of guanosine 5'-3-O-(thio)triphosphate. These data support the hypothesis that amphiphysin I, dynamin I, and synaptojanin I are...