Acidic and basic fibroblast growth factors (aFGF and bFGF) belong to a family of structurally related polypeptides characterized by a high affinity for heparin. a and bFGF display mitogenic activity for many cell types. Biological activity is strongly potentiated by heparin which stabilizes their molecular conformation by preventing physicochemical or enzymatic degradation. In our previous study we have shown that a water-soluble derivatized dextran named DDE, containing 82.2% methyl carboxylic acid groups, 6.1% benzylamide, and 5.6% sulfonate with a specific anticoagulant activity equivalent to heparin of 0.5 IU/mg could potentiate the mitogenic activity of aFGF on CCL39 cells. Optimal concentrations for maximal potentiation of 400 micrograms/ml and 20 micrograms/ml were obtained respectively for DDE and heparin. In the present report, we have uncovered the fact that several carboxymethyl benzylamide sulfonate dextrans differing in degree and positioning of the substituent groups can mimic heparin in regard to the protection, stabilization, and potentiating effects with aFGF or bFGF. Our data establishes that the dextran derivatives studied can act as potentiating agents for FGFs. Native dextran (DDA) had no effect. Dextran derivatives can also protect aFGF and bFGF from heat as well as from pH denaturation, and against trypsic and chymotrypsic degradation. The dextran derivative DDI (82% methylcarboxylic acid, 23% benzylamide, 13% sulfonate) was studied in greater detail and exhibited a greater protection for bFGF and a lesser protecting effect for aFGF than heparin. Derivatized dextrans which have very weak anticoagulant activity are of great interest as alternatives to heparin for use as stabilizers, potentiators, protectants, and slow-release matrices for FGFs in pharmaceutical formulations.

Dynamin is a GTPase involved in endocytosis and other aspects of membrane trafficking. A critical function in the presynaptic compartment attributed to the brain-specific dynamin isoform, dynamin-1, is in synaptic vesicle recycling. We report that dynamin-2 specifically interacts with members of the Shank/ProSAP family of postsynaptic density scaffolding proteins and present evidence that dynamin-2 is specifically associated with the postsynaptic density. These data are consistent with a role for this otherwise broadly distributed form of dynamin in glutamate receptor down-regulation and other aspects of postsynaptic membrane turnover.Dynamin is a 100-kDa GTPase (1, 2) that controls a variety of vesicular budding events including synaptic vesicle recycling, receptor-mediated endocytosis, caveolae internalization, phagocytosis, and secretory vesicle budding from the transGolgi network (3-9). It forms long spiral polymers around the necks of coated pits (10) and on lipid tubules (11), suggesting that the protein may directly function in membrane scission. Alternatively, dynamin has also been postulated to act as a GTPase switch by recruiting other endocytic factors to the neck and then activating them to sever the coated vesicle (12).Dynamin contains an amino-terminal GTPase domain, followed by a central coiled-coil assembly domain (13), a pleckstrin homology domain, which binds to phosphoinositides and the ␤␥ subunits of heterotrimeric GTPases (14,15), and a carboxyl-terminal coiled-coil region (also called the assembly or GTPase effector domain) that is involved in self-association (13,16,17). At the extreme carboxyl terminus is a basic, prolinerich domain to which a number of Src homology 3 (SH3) 1 domain-containing proteins, acidic phospholipids, and microtubules have been shown to bind (18 -20).Considerable insight into dynamin function at the synapse has come from genetic and morphological studies on the temperature-sensitive mutants of shibire, the dynamin ortholog in Drosophila (21,22). Single point mutations in the GTPase domain of shibire cause paralysis at elevated temperatures, and ultrastructural analysis of nerve terminals under these conditions has revealed a depletion of synaptic vesicles, along with an accumulation of collared pits (23,24).In mammals, three closely related dynamin genes are expressed in a tissue-specific manner. Dynamin-1 is almost exclusively expressed in neurons (25). Dynamin-2 is found in the brain but is also widely expressed among other tissues (26 -28). Dynamin-3 was initially identified in testis (29) but is also found in brain, lung, and heart (30). Differences in the subcellular distribution of the dynamin gene products and their alternative splice forms have been reported (30). Because of its restriction to neurons, dynamin-1 has been assumed to be the synaptic isoform. The function of dynamin-2 is less well understood, and a role in neurons has not been identified. Overexpression of a dominant inhibitory mutant form of dynamin-2 in cultured hippocampal neurons was recen...

We demonstrated previously that forced expression of the neuronal phosphoprotein neuromodulin (also known as GAP-43, F1, B-50, and p57) in mouse anterior pituitary AtT-20 cells enhances depolarization-mediated secretion and alters cellular morphology. Here we analyze the role of calmodulin binding by neuromodulin in these responses. In cells expressing wild-type neuromodulin, a complex with calmodulin that is sensitive to intracellular calcium and phosphorylation is localized to the plasma membrane. Transfection of several mutant forms of neuromodulin shows that the effects of this protein on secretion are dependent on both calmodulin binding and association with the plasma membrane. In contrast, the morphological changes depend only on membrane association. Thus, the multitude of effects of neuromodulin noted in previous studies may result from divergent properties of this protein.The neuronal growth-associated protein neuromodulin (also designated GAP-43, B-50, and F1) is a membrane-bound phosphoprotein expressed at a high level during neuronal development and regeneration (reviewed in Refs. 1 and 2). Neuromodulin is a rapidly transported axonal protein (3-10) that is concentrated in the growth cone of elongating axons (8,(11)(12)(13)(14)(15)(16)(17). Additionally, overexpression of neuromodulin in the nervous system of transgenic mice causes spontaneous nerve sprouting at the neuromuscular junction and potentiates lesion-induced nerve sprouting and terminal arborization during re-innervation (18). Taken together, these results suggest that neuromodulin plays an important role in axon elongation. Further support for this notion has been derived from studies of several cell culture model systems (19 -28).However, a line of PC12 cells in which neuromodulin expression is nearly undetectable is nevertheless capable of robust neurite elongation in response to nerve growth factor (29). Additionally, cultured neurons derived from mice in which the neuromodulin gene has been disrupted by gene targeting extend axons to the same extent as cells that express this protein.Further analysis of the neuromodulin (Ϫ) embryos revealed that retinal axons appear incapable of crossing the midline decision point in the optic chiasm, implying that their growth cones fail to respond to environmental guidance cues (30).These results suggest that neuromodulin is perhaps not essential for axon elongation, but rather might function as a mediator of signal transduction pathways in the growth cone that serve to modulate the rate, extent, and trajectory of axonal growth.In the adult nervous system, neuromodulin expression persists in pre-synaptic terminals in those regions where the synaptic modifications associated with learning and memory are thought to occur (31-36). Additionally, the correlation of PKC 1 -mediated neuromodulin phosphorylation with long term potentiation of synaptic transmission in the hippocampus (37-40) and neurotransmitter release in vitro (41) suggest a role for neuromodulin in neuronal plasticity, possibly via modul...

The neuronal growth-associated protein (GAP)-43 (neuromodulin, B-50, F1), which is concentrated in the growth cones of elongating axons during neuronal development and in nerve terminals in restricted regions of the adult nervous system, has been implicated in the release of neurotransmitter. To study the role of GAP-43 in evoked secretion, we transfected mouse anterior pituitary AtT-20 cells with the rat GAP-43 cDNA and derived stably transfected cell lines. Depolarization-mediated beta-endorphin secretion was greatly enhanced in the GAP-43-expressing AtT-20 cells without a significant change in Ca2+ influx; in contrast, expression of GAP-43 did not alter corticotropin-releasing factor-evoked hormone secretion. The transfected cells also displayed a flattened morphology and extended processes when plated on laminin-coated substrates. These results suggest that AtT-20 cells are a useful model system for further investigations on the precise biological function(s) of GAP-43.