MOLECULAR MECHANISMS IN THE REGULATION OF 
NEUROTRANSMITTER RECEPTOR FUNCTION 
Richard L. Huganir, Ph.D., Associate Investigator 
Synaptic transmission is the process by which 
neurons in the brain communicate with each other 
through chemical neurotransmitters. During sy- 
naptic transmission, an action potential in the 
presynaptic neuron triggers the release of neuro- 
transmitters from the nerve terminal; these neuro- 
transmitters then diffuse across the synaptic cleft 
and bind to neurotransmitter receptors in the post- 
synaptic membrane. The binding of the neurotrans- 
mitter to the receptor causes the opening of ion 
channels in the postsynaptic membrane, allowing 
ions to diffuse down their electrochemical gradient 
and depolarize the cell. This depolarization triggers 
an action potential in the postsynaptic cell and thus 
completes the process of synaptic transmission. 
Dr. Huganir's laboratory is interested in the mo- 
lecular mechanisms that underlie the regulation of 
synaptic transmission. Recent studies in many labo- 
ratories have provided evidence that protein phos- 
phorylation may be one of the major mechanisms 
in the control of synaptic transmission. To analyze 
the role of protein phosphorylation in the control 
of synaptic function, Dr. Huganir and his colleagues 
have used the best-characterized neurotransmitter 
receptor in neurobiology, the nicotinic acetylcho- 
line receptor, as a model system. The nicotinic ace- 
tylcholine receptor is a chemically gated ion chan- 
nel that is a pentameric complex of four types of 
subunits, in the stoichiometry a2P78. The nicotinic 
receptor mediates the response to the neurotrans- 
mitter acetylcholine at the postsynaptic membrane 
of nicotinic cholinergic synapses such as the neuro- 
muscular junction. 
L Characterization of Protein Phosphorylation of 
the Nicotinic Acetylcholine Receptor. 
Initially Dr. Huganir and his colleagues examined 
the role of protein phosphorylation in the regula- 
tion of synaptic transmission at nicotinic synapses 
by characterizing the protein kinases that phospho- 
rylate the nicotinic receptor. Postsynaptic mem- 
branes highly enriched in the nicotinic receptor 
were isolated from the electric organ of Torpedo 
californica and were found to contain at least 
three different protein kinases that phosphorylat- 
ed the nicotinic receptor. The cAMP-dependent 
protein kinase phosphorylated the receptor on the 
7- and 8-subunits, protein kinase C phosphorylated 
the 8- and a-subunits, and a protein tyrosine kinase 
activity phosphorylated the (3-, 7-, and 8-subunits of 
the receptor. Each of these kinases phosphorylated 
a unique site on each subunit; thus three different 
protein kinases phosphorylated the receptor on a 
total of seven distinct sites. All seven of these phos- 
phorylation sites are located on a homologous re- 
gion on the major intracellular loop of each sub- 
unit. In addition, multiple phosphorylation sites on 
a single subunit are clustered together; for exam- 
ple, the three phosphorylation sites on the 8-sub- 
unit are located within 20 amino acids of each 
other, suggesting that phosphorylation of the re- 
ceptor by all three protein kinases regulates a com- 
mon property of the receptor. All of these phos- 
phorylation sites have been found to be conserved 
in the amino acid sequence of acetylcholine recep- 
tor subunits from all species sequenced so far, ex- 
cept for the phosphorylation sites on the 7-sub- 
units from mammalian species. However, the 
cAMP-dependent phosphorylation site is conserved 
in the adult form of the 7-subunit (e-subunit) in 
mammalian species. 
To identify the protein tyrosine kinase (s) that 
phosphorylates the receptor. Dr. Huganir and his 
colleagues are using protein purification and mo- 
lecular cloning techniques. Immunological cross-re- 
action with known protein tyrosine kinases and 
partial purification of the protein tyrosine kinases 
have shown that there are at least two distinct pro- 
tein tyrosine kinases in Torpedo electroplax. The 60 
kDa protein tyrosine kinase is related to pp60'^*'^'^, a 
well-characterized protein tyrosine kinase. The 
other protein tyrosine kinase appears to be a novel 
90 kDa protein kinase that seems to be specific to 
the acetylcholine receptor-rich postsynaptic mem- 
branes. The 90 kDa protein has recently been par- 
tially purified, and attempts are being made to ob- 
tain the amino acid sequence of the protein to 
prepare specific oligonucleotide probes for cDNA 
cloning. In addition, polymerase chain reaction and 
cDNA cloning techniques have been used to iden- 
tify four different protein tyrosine kinases that are 
expressed in the Torpedo electric organ. These four 
different protein tyrosine kinases are being ana- 
lyzed to determine which of them is involved in the 
phosphorylation and regulation of the nicotinic 
acetylcholine receptor. 
The nicotinic acetylcholine receptor is phosphor- 
ylated in muscle cells by the same protein kinases 
Continued 
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