Molecular Mechanisms in the Regulation of Synaptic Transmission 
tyrosine kinase phosphorylates the |8-, 7-, and 
5-subunits. These postsynaptic membranes also 
contain phosphoprotein phosphatase activity 
that dephosphorylates the phosphorylated nico- 
tinic acetylcholine receptor. 
We are currently using protein purification and 
molecular cloning techniques to characterize the 
protein-tyrosine kinases that phosphorylate the 
receptor and the phosphotyrosine protein phos- 
phatases that dephosphorylate the receptor. We 
recently identified several cDNA clones for dif- 
ferent types of protein-tyrosine kinases that are 
expressed in cells enriched in the nicotinic re- 
ceptor and are attempting to determine which of 
these protein-tyrosine kinases phosphorylate the 
receptor. In addition, we recently purified the 
phosphotyrosine protein phosphatase that de- 
phosphorylates the tyrosine-phosphorylated ace- 
tylcholine receptor and are using molecular 
cloning techniques to isolate cDNA clones for 
this phosphotyrosine protein phosphatase. 
What are the functional effects of phosphoryla- 
tion of the receptor by these protein kinases? We 
have examined this question directly by studying 
the properties of the purified and reconstituted 
receptor phosphorylated to different degrees by 
the various protein kinases. Phosphorylation of 
the receptor on the 7- and 6-subunits by cAMP- 
dependent protein kinase or phosphorylation of 
the receptor on the 0-, 7-, and 5-subunits by the 
protein-tyrosine kinase dramatically increases the 
rate of desensitization of the receptor. Desensiti- 
zation is the process by which the receptor is re- 
versibly inactivated in the continued presence of 
the neurotransmitter acetylcholine. These stud- 
ies provide direct evidence that protein phos- 
phorylation of the nicotinic acetylcholine recep- 
tor regulates its physiological properties and 
plays a role in modulating its sensitivity to 
acetylcholine. 
To analyze the effect of phosphorylation on the 
desensitization of the receptor in more detail, we 
have recently used site-specific mutagenesis tech- 
niques to mutate the phosphorylation sites on the 
receptor subunits. Mutant receptor subunits lack- 
ing phosphorylation sites have been expressed in 
Xenopus oocytes, and the regulation of desensiti- 
zation of these receptors by protein phosphoryla- 
tion is being analyzed and compared with normal 
receptors. 
Using muscle cell cultures that are highly 
enriched in the acetylcholine receptor, we have 
investigated the regulation of the phosphoryla- 
tion of the receptor by neurotransmitters, hor- 
mones, and neuropeptides. Calcitonin gene- 
related peptide (CGRP), a neuropeptide that is 
released from the presynaptic neuron with ace- 
tylcholine, increases the intracellular levels of 
cAMP and thereby regulates the phosphorylation 
of the receptor by the cAMP-dependent protein 
kinase. In addition, studies in our laboratory sug- 
gest that acetylcholine itself regulates intracellu- 
lar levels of calcium and thereby regulates the 
phosphorylation of its own receptor by the cal- 
cium- and diacylglycerol-dependent protein ki- 
nase. We have also demonstrated that tyrosine 
phosphorylation of the nicotinic receptor is regu- 
lated by the neurons that synapse on muscle. 
More recently, in collaboration with Bruce Wal- 
lace (University of Colorado Health Sciences 
Center), we have found that agrin, an extracellu- 
lar matrix protein, may be the factor from neu- 
rons that regulates tyrosine phosphorylation of 
the receptor. Agrin is a well-characterized pro- 
tein that is secreted from neurons and induces 
receptor clustering under the nerve during syn- 
apse formation. These results suggest that agrin- 
induced tyrosine phosphorylation of the receptor 
may be involved in the induction of clustering of 
the receptor at the synapse. 
Our recent studies on GABA^ and glutamate re- 
ceptors have supported our hypothesis that pro- 
tein phosphorylation of neurotransmitter recep- 
tors plays an important role in the modulation of 
their function. We have expressed the genes for 
these receptors in a variety of cells and have ana- 
lyzed the effect of phosphorylation on their func- 
tional properties. Using this system, we have 
shown that phosphorylation of the /3-subunit by 
cAMP-dependent protein kinase decreases the re- 
sponse of the GABA^ receptor to its neurotransmit- 
ter and alters the desensitization of the receptor. 
In addition, recent studies have demonstrated 
that glutamate receptors are phosphorylated by a 
protein-tyrosine kinase. The functional effect of 
tyrosine phosphorylation of the glutamate recep- 
tor is currently being examined. This work, com- 
bined with our studies of the nicotinic acetylcho- 
line receptor, provides strong evidence that 
protein phosphorylation of neurotransmitter re- 
ceptors is a primary mechanism in the regulation 
of synaptic transmission. 
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