Development and Function of the Synapse 
brain, resulting in a variety of combinations of 
these molecules on different vesicles. 
Molecular fractionation and immunoprecipita- 
tion techniques suggest that several of the synap- 
tic vesicle proteins interact to form a large multi- 
meric complex. This complex contains a 
previously uncharacterized 35-kDa protein that 
is a major substrate for casein kinase. We have 
purified this protein and isolated cDNAs encod- 
ing the molecule. The protein is unique in the 
database and is predicted to be anchored in the 
membrane by a hydrophobic carboxyl terminus 
and to have an amino-terminal region oriented 
toward the cytoplasm. Like the synaptic vesicle 
proteins, this molecule is a member of a small 
gene family. Experiments are directed tow^ard un- 
derstanding the localization and function of this 
protein and the other synaptic vesicle proteins. 
Synapse Development 
Motor neurons in the spinal cord send axons to 
muscle fibers throughout the body. When axons 
contact muscle fibers, a highly ordered structure 
consisting of a presynaptic nerve terminal and a 
postsynaptic site develops. The presynaptic ter- 
minal comprises an active zone rich in synaptic 
vesicles containing neurotransmitter. The post- 
synaptic element is made up of a membrane rich 
in receptors for the neurotransmitter and an in- 
dentation in the membrane called the junctional 
fold. An extracellular matrix, or basal lamina, 
surrounds the muscle fiber, including the space 
between the nerve and muscle. 
One of the key events in the development of 
the neuromuscular junction is the redistribution 
of neurotransmitter receptors that occurs when 
nerve contacts muscle. Initially receptors for the 
neurotransmitter, in this case acetylcholine, are 
randomly distributed on the muscle fiber. When 
the nerve contacts muscle, neurotransmitter re- 
ceptors aggregate under the nerve terminal in an 
appropriate position to detect the chemicals re- 
leased during synaptic transmission. 
Agrin, a component of the extracellular matrix, 
causes acetylcholine receptors to cluster when 
added to muscle fibers growing in culture. We 
have isolated recombinant DNA clones encoding 
agrin molecules and, through an analysis of the 
nucleotide sequence, have defined the primary 
amino acid sequence of the molecule. When we 
compare the predicted agrin sequence with the 
proteins in the data bank, two types of similarities 
are revealed. The first is to a class of molecules 
that inhibit proteases and the second to a protein 
motif called EGF (epidermal growth factor) re- 
peats. The gene is expressed in embryonic motor 
neurons at the time they are first contacting mus- 
cle fibers. Two regions of the agrin gene are alter- 
nately spliced and may produce up to eight forms 
of the molecule. 
Expression of agrin encoding cDNAs in CHO 
and COS cell lines results in the association of the 
protein with the surface of the transfected cells, 
probably through assembly into an extracellular 
matrix. Coculture of agrin-expressing cells with 
primary muscle fibers or a C2 myoblast cell line 
results in aggregation of acetylcholine receptors 
at sites of contact between the transfected cell 
and the muscle fibers. Only the forms of the pro- 
tein containing an eight-amino acid sequence, 
which is the product of alternate RNA splicing, 
are capable of causing clusters on S27 cells, a 
mutant C2 line lacking proteoglycans. These data 
suggest that agrin may cluster receptors via two 
mechanisms, one of which is proteoglycan de- 
pendent. Another possibility is that the eight 
amino acids provide a binding site that results in a 
high-affinity interaction, overriding the need for 
the proteoglycan component. 
Since agrin is stably maintained in the synaptic 
basal lamina after nerve or muscle damage, it may 
also play a role in regeneration events. Under- 
standing the mechanisms of peripheral synapse 
regeneration may lead to procedures that could 
aid in central nervous system regeneration. 
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