Synaptotagmin-1 Is Required for Fibroblast Growth Factor-1 Release

Abstract

By using p65 synaptotagmin-1 and fibroblast growth factor (FGF)-1:␤-galactosidase (␤-gal) NIH 3T3 cell cotransfectants, we demonstrate that a proteolytic fragment consisting of the extravesicular domain of synaptotagmin-1 is released into the extracellular compartment in response to temperature stress with similar kinetics and pharmacological properties as FGF-1:␤-gal. Using a deletion mutant that lacks 95 amino acids from the extravesicular domain of synaptotagmin-1, neither synaptotagmin-1 nor FGF-1:␤-gal are able to access the stress-induced release pathway. Furthermore, the p40 extravesicular fragment of synaptotagmin-1 is constitutively released in p40 synaptotagmin-1 NIH 3T3 cell transfectants, and this release is potentiated when the cells are subjected to temperature stress. These data demonstrate that the p40 fragment derived from synaptotagmin-1 is able to utilize the FGF-1 non-classical exocytotic pathway and that the release of FGF-1 is dependent on synaptotagmin-1. FGF1 -1 and FGF-2 are the prototype members of a large family of related heparin-binding growth factors that regulate numerous biological processes including mesoderm formation, neurogenesis, and angiogenesis in vivo (1). The FGF prototypes are distinguished among the FGF family members by the absence of a classical signal peptide sequence to direct their secretion through the conventional endoplasmic reticulum (ER)-Golgi pathway. Because the FGF prototypes initiate their biological responses through the activation of high affinity receptors on the surface of target cells, it has been suggested that the secretion of both FGF-1 and FGF-2 may be regulated by novel release pathways. Indeed, the ligation of a signal peptide sequence to FGF-1 followed by somatic gene transfer, which results in the constitutive release of FGF-1, yields a functional oncogene in vitro and exaggerated vascular hyperplasia in vivo (2, 3).We have reported that FGF-1 is released from stable FGF-1 NIH 3T3 cell transfectants through a non-classical exocytotic pathway as a latent macromolecule in response to temperature stress which requires activation by either (NH 4 ) 2 SO 4 (4) or reducing agents (5-7). During the structural characterization of FGF-1 (8), a heparin-binding protein (p40) that co-eluted with FGF-1 was identified by amino-terminal sequence analysis as a proteolytic fragment of the synaptotagmin-1 (Syn-1) translation product (8). Syn-1 is a 65-kDa vesicular protein and has been most extensively studied in neuronal and endocrine cells. The Syn gene family currently consists of 11 members (9, 10) and is characterized by a divergent amino terminus, a transmembrane domain, a large cytoplasmic region, which is comprised of two repeats of sequences homologous to the protein kinase C C2 domain, and a highly conserved carboxyl terminus (11,12). The p40 proteolytic fragment identified in neural tissue corresponds to the extravesicular portion of Syn-1. Genetic and microinjection studies have suggested that Syn-1 is the Ca 2ϩ sensor for neurotransmitter release (1...

Release of FGF1 and p40 synaptotagmin 1 correlates with their membrane destabilizing ability

“…The plasmids for eukaryotic expression of FGF1 (pXZ38) and p40 Syt1 (p40-Syt1:Myc in pMEX hygro) were prepared as previously described [14,21,22]. The K326,327,331Q mutant of p40 Syt1 was produced by mutagenesis from the p40 Syt1:Myc pMEX Hygro and GST-p40 Syt1-pGEX-KG original plasmids [22,34] for eukaryotic and prokayotic expression, respectively.…”

“…The K326,327,331Q mutant of p40 Syt1 was produced by mutagenesis from the p40 Syt1:Myc pMEX Hygro and GST-p40 Syt1-pGEX-KG original plasmids [22,34] for eukaryotic and prokayotic expression, respectively. FGF1 K126,127A and FGF1 K114,115A mutants were produced by mutagenesis of the FGF1:HA pCR3.1 construct (gift of Andrew Baird, Human BioMolecular Research Institute, San Diego, CA) for eukaryotic expression, and the FGF1 pET3C construct [35] for prokaryotic expression.…”

“…Transiently transfected NIH 3T3 cells grown to 70-80% confluency were washed with DMEM containing 5 units/ml heparin (Sigma), and heat shock was performed as previously described [14] in DMEM containing 5 units/ml heparin for 110 minutes at 42°C; control cultures were incubated at 37°C in the same medium. Released p40 Syt1 was isolated using heparin Sepharose chromatography as described [22]. Released FGF1:HA was immunoprecipitated using anti-HA monoclonal antibodies (Covance) as described [21].…”

“…FGF1 is secreted from cells upon stress stimulation such as heat shock [14], hypoxia [17], serum starvation [18], and treatment with oxidized LDL [19]. The availability of free intracellular copper ions is necessary for FGF1 release, and in vitro data suggest the formation of a copper-and stress-dependent multiprotein export complex [20], which contains the calcium-binding proteins, S100A13 and p40 Syt1 [21,22]. p40 Syt1 is a non-transmembrane isoform of the integral component of secretory vesicles, synaptotagmin (Syt)1, that is involved in the fusion of exocytotic vesicles with the plasma membrane [23].…”

“…Their constitutive release is blocked when they are cotransfected into the cells along with FGF1; however upon stress, they are released in a complex with FGF1 [21,22].…”