Analysis of the Role of Calmodulin Binding and Sequestration in Neuromodulin (GAP-43) Function

Abstract

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...

Molecular Mechanisms, Biological Actions, and Neuropharmacology of the Growth-Associated Protein GAP-43

“…However, since the binding of GAP-43 to calmodulin is terminated upon the phosphorylation of GAP-43 by protein kinase C, it implies that the GAP-43-calmodulin interaction occurs mainly in the resting cell. Consistent with this idea, a crosslinking study showed that GAP-43 is bound to calmodulin in vivo under conditions of low calcium but is released when the calcium concentration is increased or when GAP-43 is phosphorylated by protein kinase C [33].…”

An α7 nicotinic receptor-G protein pathway complex regulates neurite growth in neural cells*

“…To determine if change in GAP-43 phosphorylation correlated with CaM binding, we utilized the bis(sulfosuccinimidyl) substrate (BS 3 ) to test the effects of Bgtx and Nic on GAP-43/CaM interaction. This method has been used in the detection of GAP-43 in the CaM bound/free states where the GAP-43 band runs at ,18 kDa (the approximate size of CaM) heavier on an SDS-PAGE gel when bound to CaM (Gamby et al, 1996). In these experiments, cell fractions were crosslinked following a 2-hour treatment with Bgtx, Nic, or a vehicle control.…”

Interactions between Neurogranin and Calmodulin in Vivo

“…Therefore, it has been important to determine whether these proteins bind CaM and are phosphorylated by PKC in vivo. Both neurogranin and neuromodulin are phosphorylated by PKC in vivo (8,43,44), and recently it was shown that neuromodulin binds CaM in vivo (45,46). The objectives of this study were to determine if neurogranin binds CaM in vivo through its IQ domain and to determine if other neurograninbinding proteins can be detected by yeast two-hybrid technology.…”