Abstract
It has been suggested that GAP-43 (growth-associated protein) binds to various proteins in growing neurons as part of its mechanism of action. To test this hypothesis in vivo, differentiated N1E-115 neuroblastoma cells were labeled with [35S]-amino acids and were treated with a cleavable crosslinking reagent. The cells were lysed in detergent and the lysates were centrifuged at 100,000 × g to isolate crosslinked complexes. Following cleavage of the crosslinks and analysis by two-dimensional gel electrophoresis, it was found that the crosslinker increased the level of various proteins, and particularly actin, in this pellet fraction. However, GAP-43 was not present, suggesting that GAP-43 was not extensively crosslinked to proteins of the cytoskeleton and membrane skeleton and did not sediment with them. GAP-43 also did not sediment with the membrane skeleton following nonionic detergent lysis. Calmodulin, but not actin or other proposed interaction partners, co-immunoprecipitated with GAP-43 from the 100,000 × g supernatant following crosslinker addition to cells or cell lysates. Faint spots at 34 kDa and 60 kDa were also present. Additional GAP-43 was recovered from GAP-43 immunoprecipitation supernatants with anti-calmodulin but not with anti-actin. The results suggest that GAP-43 is not present in complexes with actin or other membrane skeletal or cytoskeletal proteins in these cells, but it is nevertheless possible that a small fraction of the total GAP-43 may interact with other proteins.
Highlights
The growth-associated protein growth-associated protein of 43 kDa (GAP-43) participates in neuronal development and is present in certain regions of the normal adult brain, retina, and cornea [1,2,3,4]
The results suggest that the majority of GAP-43 in growing N1E-115 cells is not complexed to other proteins, but a small fraction of the total
The mouse neuroblastoma cell line N1E-115 was differentiated by the addition of 2% dimethyl sulfoxide (DMSO) to the medium
Summary
The growth-associated protein GAP-43 participates in neuronal development and is present in certain regions of the normal adult brain, retina, and cornea [1,2,3,4]. Proteasome digestion studies suggested that GAP-43 is primarily unfolded at physiological ionic strength [7] It is of interest how the protein might fold following its interaction with the membrane and with other proteins. GAP-43 binds to calmodulin in vitro [23], in synaptosomes [24], and in vivo [25] This interaction is eliminated following the phosphorylation of GAP-43 by protein kinase C [23, 25]. The addition of a membrane permeable crosslinker to living, stimulated neurons is one approach to test for the direct interaction of GAP-43 with other proteins. It was found that the crosslinker caused calmodulin, but not actin or other proposed interaction partners, to co-immunoprecipitate with GAP-43. GAP-43 could be involved in some protein-protein interactions
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