MOLECULAR BIOLOGY OF NERVE TERMINAL PLASTICITY 
Mark C. Fishman, M.D., Associate Investigator 
Regulated growth and retraction of nerve termi- 
nals mediate changes in neuronal connectivity dur- 
ing development, repair, and memory. Dr. Fishman 
and his colleagues are investigating the molecular 
basis of this plasticity. Over the recent years they 
have focused on the gene for the GAP-43 protein, 
since this gene is regulated concordantly with 
growth and the protein is enriched in growth cone 
membranes. 
I. GAP-43 and the Growth Cone. 
The previously cloned GAP-43 cDNA has been 
used to isolate and analyze the rat gene for GAP-43. 
Ed Grabczyk and Dr. Mauricio Zuber have deter- 
mined some elements of the GAP-43 promoter: it 
lacks TATA or CAAT boxes, has multiple transcrip- 
tional start sites, and includes a region of H, or a 
triple-stranded, DNA. Adjacent to this region is a 
motif identical to that recognized by transcriptional 
regulators of the POU family. Dr. Sally Teng and 
Dr. Zuber have cloned a brain-specific protein that 
binds to this region. They are evaluating the role 
of the promoter's structure and this trans-acting 
protein in GAP-43 gene regulation. Other regions 
that are subject to regulation by, for example, corti- 
costeroids or nerve growth factor (NGF), reside 
within introns or the 3 -untranslated region of the 
gene. 
The function of GAP-43 is not known, although it 
has been speculated that it plays a role in nerve ter- 
minal plasticity. To establish a bioassay for GAP-43, 
in isolation from endogenous GAP-43 and other 
neuronal proteins. Dr. Zuber generated nonneuro- 
nal cell lines that express GAP-43 at high levels. In 
this context the expression of GAP-43 causes 
marked filopodial extension from the cell surface. 
Because these cells lack specific neuronal ma- 
chinery, it is likely that GAP-43 can interact with 
structural components that are more universally ex- 
pressed and that normally cause cell shape changes 
for mitosis or migration. This work suggests that 
the role of GAP-43 may be to regulate filopodial ex- 
tension and permits mutational analysis of the re- 
gions of GAP-43 that are important to this pheno- 
type. This assay is also being used to identify other 
molecular components that interact with GAP-43 to 
cause membrane remodeling. 
In an attempt to understand these other pans of 
the growth cone transduction system. Dr. Stephen 
Strittmatter has isolated the major protein compo- 
nents of growth cone membranes. Two of the most 
prominent are the a- and (B-subunits of G^. Their 
specific subtypes were identified by microsequenc- 
ing, in collaboration with Dr. Tim Kennedy. The G 
protein family functions to link membrane recep- 
tors to intracellular signaling systems, but the role 
of G^, in particular, is poorly defined. Its growth 
cone enrichment suggests a role in that structure. 
Dr. Fishman's laboratory has also shown that GAP- 
43 and G^ are physically associated. The notion 
that a complex of proteins (including GAP-43 and 
G^) constitute a chemical/mechanical transduction 
system that converts extracellular stimuli into di- 
rected growth is being tested. 
GAP-43, although tightly associated with the 
growth cone membrane, bears no transmembrane 
domain in its structure. This suggests that it con- 
tains a domain responsible for enrichment in one 
particular region of the plasma membrane, the 
growth cone. This would be akin to "sorting se- 
quences," short stretches of amino acids that deter- 
mine whether proteins in epithelial cells accumu- 
late on the basal or apical membranes. Drs. Zuber 
and Strittmatter have studied this possibility by mu- 
tational analysis and generation of chimeric pro- 
teins between pieces of GAP-43 and the cytosolic 
protein chloramphenicol acetyltransferase (CAT). 
The amino terminus of GAP-43 has been identified 
as responsible for membrane binding. This region 
causes chimeric proteins to accumulate in a distri- 
bution indistinguishable from that of GAP-43, 
which in neuronal cell lines includes the growth 
cone. The two amino-terminal cysteines are espe- 
cially critical to this guidance. The possibility that 
accumulation occurs because the amino terminus 
binds to other proteins, perhaps GAP-43 "recep- 
tors" on the inner face of the plasma membrane, is 
being investigated. These studies suggest that GAP- 
43 bears a cellular trafficking domain that guides it 
to specific regions of the cell, especially those 
where remodeling occurs, where it interacts with 
membrane transduction systems to enhance 
filopodial extension. 
II. Transgenic Models of Sensory Neuropathy. 
Although it would be of great interest to evalu- 
ate the effects of disturbing gene expression in neu- 
rons, especially in their normal environment, it is 
Continued 
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