Amphiphysin I Is Associated with Coated Endocytic Intermediates and Undergoes Stimulation-dependent Dephosphorylation in Nerve Terminals
1997; Elsevier BV; Volume: 272; Issue: 49 Linguagem: Inglês
10.1074/jbc.272.49.30984
ISSN1083-351X
AutoresRudolf Bauerfeind, Kohji Takei, Pietro De Camilli,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoAmphiphysin 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 μm), two drugs that specifically block the Ca2+/calmodulin-dependent phosphatase 2B calcineurin, but not by okadaic acid (1 μm), 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 physiological partners in some step(s) of synaptic vesicle endocytosis. We hypothesize that the parallel Ca2+-dependent calcineurin-dependent dephosphorylation of amphiphysin I and of its two major binding proteins is part of a process that primes the nerve terminal for endocytosis in response to a burst of exocytosis. 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 μm), two drugs that specifically block the Ca2+/calmodulin-dependent phosphatase 2B calcineurin, but not by okadaic acid (1 μm), 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 physiological partners in some step(s) of synaptic vesicle endocytosis. We hypothesize that the parallel Ca2+-dependent calcineurin-dependent dephosphorylation of amphiphysin I and of its two major binding proteins is part of a process that primes the nerve terminal for endocytosis in response to a burst of exocytosis. Amphiphysin I, an autoantigen in neurological autoimmune paraneoplastic conditions (1Folli F. Solimena M. Cofiell R. Austoni M. Tallini G. Fassetta G. Bates D. Cartlidge N. Bottazzo G.F. Piccolo G. et al.N. Engl. J. Med. 1993; 328: 546-551Crossref PubMed Scopus (272) Google Scholar, 2De Camilli P. Thomas A. Cofiell R. Folli F. Lichte B. Piccolo G. Meinck H.M. Austoni M. Fassetta G. Bottazzo G. Bates D. Cartlidge N. Solimena M. Kiliman M.W. J. Exp. Med. 1993; 178: 2219-2223Crossref PubMed Scopus (274) Google Scholar), is a cytosolic protein expressed at high levels in the nervous system where it is concentrated in presynaptic nerve terminals (3Lichte B. Veh R.W. Meyer H.E. Kilimann M.W. EMBO J. 1992; 11: 2521-2530Crossref PubMed Scopus (164) Google Scholar, 4David C. McPherson P.S. Mundigl O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar). It contains an NH2-terminal coiled-coil domain and a COOH-terminal src homology 3 (SH3) 1The abbreviations used are: SH3, src homology 3; ΔCnA, recombinant calcineurin A lacking the autoinhibitory and calmodulin-binding domains; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; GTPγS, guanosine 5′-3-O-(thio)triphosphate; ATPγS, adenosine 5′-O-(thiotriphosphate). 1The abbreviations used are: SH3, src homology 3; ΔCnA, recombinant calcineurin A lacking the autoinhibitory and calmodulin-binding domains; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; GTPγS, guanosine 5′-3-O-(thio)triphosphate; ATPγS, adenosine 5′-O-(thiotriphosphate). domain (5David C. Solimena M. De Camilli P. FEBS Lett. 1994; 351: 73-79Crossref PubMed Scopus (130) Google Scholar,6Sivadon P. Bauer F. Aigle M. Crouzet M. Mol. Gen. Genet. 1995; 246: 485-495Crossref PubMed Scopus (113) Google Scholar). Via the SH3 domain, it interacts with the COOH-terminal proline-rich tail of two other abundant nerve terminal proteins, the GTPase dynamin I (4David C. McPherson P.S. Mundigl O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar) and the inositol-5-phosphatase synaptojanin I (7McPherson P.S. Garcia E.P. Slepnev V.I. David C. Zhang X.M. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar,8Nemoto Y. Arribas M. Haffner C. De Camilli P. J. Biol. Chem. 1997; (in press)Google Scholar). Dynamin I, which forms rings around the stalk of clathrin-coated pits (9Takei K. McPherson P.S. Schmid S.L. De Camilli P. Nature. 1995; 374: 186-190Crossref PubMed Scopus (650) Google Scholar), plays a crucial role in the endocytosis of synaptic vesicle membranes (reviewed in Refs. 10McClure S.J. Robinson P.J. Mol. Membr. Biol. 1996; 13: 189-215Crossref PubMed Scopus (74) Google Scholar, 11Urrutia R. Henley J.R. Cook T. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 377-384Crossref PubMed Scopus (253) Google Scholar, 12Cremona O. De Camilli P. Curr. Opin. Neurobiol. 1997; 7: 323-330Crossref PubMed Scopus (194) Google Scholar), and synaptojanin I may also be implicated in this process (7McPherson P.S. Garcia E.P. Slepnev V.I. David C. Zhang X.M. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar). Via a distinct domain, amphiphysin I was reported to interact with the appendage domain of the plasma membrane clathrin adaptor AP2 (4David C. McPherson P.S. Mundigl O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar, 13Wang L.-H. Südhof T.C. Anderson R.G.W. J. Biol. Chem. 1995; 270: 10079-10083Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). It has therefore been suggested that amphiphysin I may be implicated in the control of synaptic vesicle endocytosis. This possibility was recently supported by experiments carried out at the giant reticulospinal synapse of the lamprey (14Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (397) Google Scholar). Injection in the presynaptic compartment of peptides that disrupt SH3-mediated interactions of dynamin, including the interaction with amphiphysin I, was shown to produce a potent inhibition of synaptic vesicle endocytosis at the stage of invaginated clathrin-coated pits. Furthermore, the SH3 domain of amphiphysin I inhibits receptor-mediated endocytosis when transfected in fibroblastic cells (64Wigge P. Vallis Y. McMahon T. Curr. Biol. 1997; 7: 554-560Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar).A role of amphiphysin I in endocytosis is further suggested by genetic studies in the yeast Saccharomyces cerevisiae. Disruption of the genes encoding the homologue of amphiphysin, Rvs167, or the closely related protein Rvs161, produces a potent inhibition both of receptor-mediated and fluid phase endocytosis (15Munn A.L. Stevenson B.J. Geli M.I. Riezman H. Mol. Biol. Cell. 1995; 6: 1721-1742Crossref PubMed Scopus (279) Google Scholar). Mutation of theRVS161 and RVS167 genes also produces other pleotropic effects (16Crouzet M. Urdaci M. Dulau L. Aigle M. Yeast. 1991; 7: 727-743Crossref PubMed Scopus (106) Google Scholar, 17Desfarges L. Durrens P. Juguelin H. Cassagne C. Bonneu M. Aigle M. Yeast. 1993; 9: 267-277Crossref PubMed Scopus (83) Google Scholar, 18Revardel E. Bonneau M. Durrens P. Aigle M. Biochim. Biophys. Acta. 1995; 1263: 261-265Crossref PubMed Scopus (57) Google Scholar) including actin defects (6Sivadon P. Bauer F. Aigle M. Crouzet M. Mol. Gen. Genet. 1995; 246: 485-495Crossref PubMed Scopus (113) Google Scholar, 19Bauer F. Urdaci M. Aigle M. Crouzet M. Mol. Cell. Biol. 1993; 13: 5070-5084Crossref PubMed Scopus (156) Google Scholar). The latter phenotype is consistent with the general link between actin function and endocytosis, which has emerged from genetic studies in yeast (15Munn A.L. Stevenson B.J. Geli M.I. Riezman H. Mol. Biol. Cell. 1995; 6: 1721-1742Crossref PubMed Scopus (279) Google Scholar,20Benedetti H. Raths S. Crausaz F. Riezman H. Mol. Biol. Cell. 1994; 5: 1023-1037Crossref PubMed Scopus (237) Google Scholar, 21Mulholland J. Preuss D. Moon A. Wong A. Drubin D. Botstein D. J. Cell Biol. 1994; 125: 381-391Crossref PubMed Scopus (297) Google Scholar, 22Amberg D.C. Basart E. Botstein D. Nat. Struct. Biol. 1995; 2: 28-35Crossref PubMed Scopus (181) Google Scholar).A variety of nerve terminal proteins that function in neurosecretion are regulated by phosphorylation and dephosphorylation. Many of these proteins undergo an increase in phosphorylation after stimulation of neurotransmitter release (23Wang J.K. Walaas S.I. Greengard P. J. Neurosci. 1988; 8: 281-288Crossref PubMed Google Scholar). Two major nerve terminal proteins that are known to undergo a stimulation-dependent dephosphorylation (23Wang J.K. Walaas S.I. Greengard P. J. Neurosci. 1988; 8: 281-288Crossref PubMed Google Scholar, 24Robinson P.J. Hauptschein R. Lovenberg W. Dunkley P.R. J. Neurochem. 1987; 48: 187-195Crossref PubMed Scopus (41) Google Scholar) were identified as dynamin I (25Robinson P.J. Sontag J.M. Liu J.P. Fykse E.M. Slaughter C. McMahon H. Südhof T.C. Nature. 1993; 365: 163-166Crossref PubMed Scopus (235) Google Scholar) and synaptojanin I (8Nemoto Y. Arribas M. Haffner C. De Camilli P. J. Biol. Chem. 1997; (in press)Google Scholar, 26McPherson P.S. Takei K. Schmid S.L. De Camilli P. J. Biol. Chem. 1994; 269: 30132-30139Abstract Full Text PDF PubMed Google Scholar). Because amphiphysin I contains many putative phosphorylation sites (3Lichte B. Veh R.W. Meyer H.E. Kilimann M.W. EMBO J. 1992; 11: 2521-2530Crossref PubMed Scopus (164) Google Scholar, 5David C. Solimena M. De Camilli P. FEBS Lett. 1994; 351: 73-79Crossref PubMed Scopus (130) Google Scholar), we investigated whether amphiphysin I as well is regulated by phosphorylation in nerve terminals. We report here that amphiphysin I is a phosphoprotein that undergoes stimulationdependent dephosphorylation in parallel with its binding proteins, dynamin I and synaptojanin I. We also show by electron microscopy that amphiphysin I partially colocalizes with dynamin I on endocytic membrane intermediates. These findings provide new evidence for the hypothesis that amphiphysin I participates in some aspects of synaptic vesicle endocytosis.DISCUSSIONThe results of this study demonstrate that in the nerve terminal amphiphysin I undergoes constitutive phosphorylation and that stimulation by depolarization produces a decrease of its state of phosphorylation. This dephosphorylation and its pharmacological properties strikingly parallel the stimulation-dependent dephosphorylation of its two binding proteins, dynamin I (25Robinson P.J. Sontag J.M. Liu J.P. Fykse E.M. Slaughter C. McMahon H. Südhof T.C. Nature. 1993; 365: 163-166Crossref PubMed Scopus (235) Google Scholar) and synaptojanin I (26McPherson P.S. Takei K. Schmid S.L. De Camilli P. J. Biol. Chem. 1994; 269: 30132-30139Abstract Full Text PDF PubMed Google Scholar). The dephosphorylation of all three proteins is dependent on Ca2+ influx and is inhibited by inhibitors of the Ca2+/calmodulin-dependent phosphatase calcineurin. As previously shown for dynamin I (25Robinson P.J. Sontag J.M. Liu J.P. Fykse E.M. Slaughter C. McMahon H. Südhof T.C. Nature. 1993; 365: 163-166Crossref PubMed Scopus (235) Google Scholar, 44Robinson P.J. Liu J.P. Powell K.A. Fykse E.M. Südhof T.C. Trends Neurosci. 1994; 17: 348-353Abstract Full Text PDF PubMed Scopus (115) Google Scholar), the dephosphorylation of amphiphysin I is reversed by repolarization. The stimulation-dependent dephosphorylation of amphiphysin I, dynamin I, and synaptojanin I strikingly contrasts with the stimulation-dependent increase in the state of phosphorylation observed for several other nerve terminal phosphoproteins (23Wang J.K. Walaas S.I. Greengard P. J. Neurosci. 1988; 8: 281-288Crossref PubMed Google Scholar) including synapsin I and II, two proteins implicated in the exocytic branch of the synaptic vesicle cycle (37De Camilli P. Benfenati F. Valtorta F. Greengard P. Annu. Rev. Cell Biol. 1990; 6: 433-460Crossref PubMed Scopus (250) Google Scholar). These observations add further support to the hypothesis that dynamin I, synaptojanin I, and amphiphysin I are physiological partners in the endocytic reaction (12Cremona O. De Camilli P. Curr. Opin. Neurobiol. 1997; 7: 323-330Crossref PubMed Scopus (194) Google Scholar).This hypothesis is further corroborated by results of electron microscopy immunocytochemistry. Amphiphysin I was previously shown by immunofluorescence and by immunoperoxidase to be concentrated in nerve terminals (1Folli F. Solimena M. Cofiell R. Austoni M. Tallini G. Fassetta G. Bates D. Cartlidge N. Bottazzo G.F. Piccolo G. et al.N. Engl. J. Med. 1993; 328: 546-551Crossref PubMed Scopus (272) Google Scholar, 3Lichte B. Veh R.W. Meyer H.E. Kilimann M.W. EMBO J. 1992; 11: 2521-2530Crossref PubMed Scopus (164) Google Scholar, 4David C. McPherson P.S. Mundigl O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar), and by subcellular fractionation to be associated with, but not enriched on, synaptic vesicle membranes (3Lichte B. Veh R.W. Meyer H.E. Kilimann M.W. EMBO J. 1992; 11: 2521-2530Crossref PubMed Scopus (164) Google Scholar). Using the higher resolution afforded by immunogold, we have now analyzed the localization of amphiphysin I on nerve terminal membranes incubated under conditions that enhance formation of endocytic intermediates. We show that the protein is partially associated with clathrin-coated membranes and dynamin-coated membrane tubules. On both structures, amphiphysin I has a scattered distribution. Thus, amphiphysin I is not a structural partner of dynamin within dynamin rings. The similar sparse distribution of amphiphysin I and dynamin I on clathrin lattices fits with the proposal (4David C. McPherson P.S. Mundigl O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar, 14Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (397) Google Scholar, 45Okamoto P.M. Herskovits J.S. Vallee R.B. J. Biol. Chem. 1997; 272: 11629-11635Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) that the interaction of the two proteins on clathrin coats may participate in the recruitment of dynamin I, which eventually leads to its oligomerization at the neck of the vesicle bud. This hypothesis is strongly supported by peptide injection studies (14Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (397) Google Scholar) at the giant synapse of lamprey. Disruption of the interaction of dynamin I with amphiphysin I (and possibly other SH3 domain-containing proteins) at this synapse was found to produce a potent block of synaptic vesicle endocytosis at the level of invaginated clathrin-coated pits. The necks of these pits were not surrounded by a dynamin ring, as if dynamin recruitment or oligomerization had been impaired (14Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (397) Google Scholar).Amphiphysin I is homologous to the yeast protein Rvs167 (5David C. Solimena M. De Camilli P. FEBS Lett. 1994; 351: 73-79Crossref PubMed Scopus (130) Google Scholar), which has been implicated both in endocytosis and in actin function (6Sivadon P. Bauer F. Aigle M. Crouzet M. Mol. Gen. Genet. 1995; 246: 485-495Crossref PubMed Scopus (113) Google Scholar, 15Munn A.L. Stevenson B.J. Geli M.I. Riezman H. Mol. Biol. Cell. 1995; 6: 1721-1742Crossref PubMed Scopus (279) Google Scholar, 16Crouzet M. Urdaci M. Dulau L. Aigle M. Yeast. 1991; 7: 727-743Crossref PubMed Scopus (106) Google Scholar, 17Desfarges L. Durrens P. Juguelin H. Cassagne C. Bonneu M. Aigle M. Yeast. 1993; 9: 267-277Crossref PubMed Scopus (83) Google Scholar, 18Revardel E. Bonneau M. Durrens P. Aigle M. Biochim. Biophys. Acta. 1995; 1263: 261-265Crossref PubMed Scopus (57) Google Scholar, 19Bauer F. Urdaci M. Aigle M. Crouzet M. Mol. Cell. Biol. 1993; 13: 5070-5084Crossref PubMed Scopus (156) Google Scholar). Both amphiphysin I and dynamin I immunoreactivities were detectable in the cytoskeletal matrix of nerve terminals. The abundance of this immunoreactive material suggests that it does not simply represent pools of amphiphysin I and dynamin I associated with coat structures grazing into the section. This observation raises the possibility that amphiphysin I may interact with the actin cytoskeleton and suggests a link between amphiphysin I function and actin even in mammalian cells (see also Ref. 65Mundigl O. Ochoa G.C. David C. Slepnev V.I. Cabana A. De Camilli P. J. Neurosci. 1997; (in press)Google Scholar).An analysis of the amphiphysin I sequence reveals the presence of several potential phosphorylation sites for protein kinase C and casein kinase II both in the NH2-terminal coiled-coil region and in the COOH-terminal region. Our results are consistent with multisite phosphorylation of amphiphysin I and strongly suggest that calcineurin dephosphorylates the site(s) implicated in the striking electrophoretic shift of the protein. The phosphorylation-dephosphorylation of this site(s) is likely to be mediated by a cytosolic kinase in nerve terminals because it is regulated by depolarization and because the amphiphysin I doublet is enriched in synaptosomal preparations.The physiological significance of the changes in the phosphorylation state of amphiphysin I remains to be clarified. Both the upper and lower amphiphysin I bands were affinity purified by the recombinant proline-rich regions of dynamin I and synaptojanin I (data not shown). Furthermore, both bands were recovered in particulate and cytosolic fractions. Effects of phosphorylation at specific phosphorylation sites on amphiphysin I interactions need to be further investigated. Because the parallel dephosphorylations of amphiphysin I, dynamin I, and synaptojanin I occur in response to a stimulus that triggers exocytosis, and therefore compensatory endocytosis, these covalent modifications are likely to favor the endocytic reaction. In fact, dephosphorylation of dynamin I was reported to correlate with an enhanced recovery in particulate fractions (25Robinson P.J. Sontag J.M. Liu J.P. Fykse E.M. Slaughter C. McMahon H. Südhof T.C. Nature. 1993; 365: 163-166Crossref PubMed Scopus (235) Google Scholar) (possibly reflecting dynamin assembly) and with a decrease in the GTPase activity (46Liu J.P. Powell K.A. Südhof T.C. Robinson P.J. J. Biol. Chem. 1994; 269: 21043-21050Abstract Full Text PDF PubMed Google Scholar) (thus enhancing the proportion of GTP-dynamin). The state of phosphorylation of the clathrin adaptor AP2, which also binds to amphiphysin I (4David C. McPherson P.S. Mundigl O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar, 13Wang L.-H. Südhof T.C. Anderson R.G.W. J. Biol. Chem. 1995; 270: 10079-10083Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), but at a site distinct from the SH3 domain (4David C. McPherson P.S. Mundigl O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar), was not investigated in this study. However, it is of interest that AP2 assembly into coat structures was found to correlate with a decreased level of its state of phosphorylation (47Wilde A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google Scholar). Clearly, amphiphysin I dephosphorylation is an event that occurs upstream rather than downstream to the endocytic reaction because it occurs even when synaptic vesicle exocytosis (and therefore compensatory endocytosis) is blocked by tetanus toxin.The sensitivity of amphiphysin I dephosphorylation to calcineurin inhibitors provides new evidence for a regulatory role of this protein phosphatase in synaptic vesicle recycling. Although several studies have suggested a role of calcineurin in the regulation of the synaptic vesicle cycle, results have been contradictory (38Nichols R.A. 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Brodin L. Soc. Neurosci. Abstr. 1995; 21: 332Google Scholar). The parallel dephosphorylation of amphiphysin I, dynamin I, and synaptojanin I by the calcium/calmodulin-regulated protein phosphatase calcineurin may be one of the steps mediating the regulatory effects of Ca2+on synaptic vesicle endocytosis and may prime the nerve terminal for endocytic activity after a Ca2+-induced burst of exocytosis. Amphiphysin I, an autoantigen in neurological autoimmune paraneoplastic conditions (1Folli F. Solimena M. Cofiell R. Austoni M. Tallini G. Fassetta G. Bates D. Cartlidge N. Bottazzo G.F. Piccolo G. et al.N. Engl. J. Med. 1993; 328: 546-551Crossref PubMed Scopus (272) Google Scholar, 2De Camilli P. Thomas A. Cofiell R. Folli F. Lichte B. Piccolo G. Meinck H.M. Austoni M. Fassetta G. Bottazzo G. Bates D. Cartlidge N. Solimena M. Kiliman M.W. J. Exp. Med. 1993; 178: 2219-2223Crossref PubMed Scopus (274) Google Scholar), is a cytosolic protein expressed at high levels in the nervous system where it is concentrated in presynaptic nerve terminals (3Lichte B. Veh R.W. Meyer H.E. Kilimann M.W. EMBO J. 1992; 11: 2521-2530Crossref PubMed Scopus (164) Google Scholar, 4David C. McPherson P.S. Mundigl O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar). It contains an NH2-terminal coiled-coil domain and a COOH-terminal src homology 3 (SH3) 1The abbreviations used are: SH3, src homology 3; ΔCnA, recombinant calcineurin A lacking the autoinhibitory and calmodulin-binding domains; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; GTPγS, guanosine 5′-3-O-(thio)triphosphate; ATPγS, adenosine 5′-O-(thiotriphosphate). 1The abbreviations used are: SH3, src homology 3; ΔCnA, recombinant calcineurin A lacking the autoinhibitory and calmodulin-binding domains; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; GTPγS, guanosine 5′-3-O-(thio)triphosphate; ATPγS, adenosine 5′-O-(thiotriphosphate). domain (5David C. Solimena M. De Camilli P. FEBS Lett. 1994; 351: 73-79Crossref PubMed Scopus (130) Google Scholar,6Sivadon P. Bauer F. Aigle M. Crouzet M. Mol. Gen. Genet. 1995; 246: 485-495Crossref PubMed Scopus (113) Google Scholar). 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