Molecular Mechanisms in the Regulation of Synaptic Transmission 
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 dramatically increases 
the rate of desensitization of the receptor. Desen- 
sitization is the process by which the receptor is 
reversibly inactivated in the continued presence 
of the neurotransmitter acetylcholine. Recently 
we have extended these studies to examine the 
effect of phosphorylation of the receptor on the 
(8-, 7-, and 6-subunits by the protein tyrosine ki- 
nase. Phosphorylation of the receptor by the pro- 
tein tyrosine kinase also regulates the rate of de- 
sensitization of the receptor. These studies 
provide direct evidence that protein phosphory- 
lation of the nicotinic acetylcholine recep- 
tor regulates its physiological properties and 
plays a role in modulating its sensitivity to 
acetylcholine. 
We have also used site-specific mutagenesis 
techniques to mutate the phosphorylation sites 
on the receptor subunits. Mutant receptor sub- 
units lacking phosphorylation sites have been ex- 
pressed in Xenopus oocytes, in collaboration 
with Gary Yellen (HHMI, the Johns Hopkins Uni- 
versity School of Medicine) . The desensitization 
and regulation of desensitization of these recep- 
tors by protein phosphorylation are now 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 appears to be secreted from 
neurons and to induce receptor clustering under 
the nerve during synapse formation. These results 
suggest that agrin-induced tyrosine phosphoryla- 
tion of the receptor may be involved in the induc- 
tion of clustering of the receptor at the synapse. 
Our recent studies of the glutamate receptor 
and the GABA^ receptor have paralleled our stud- 
ies of the nicotinic receptor. We are using protein 
purification, reconstitution, and site-specific 
mutagenesis techniques to determine the compo- 
nents required for neurotransmitter receptor 
function and to study the role of protein phos- 
phorylation in the regulation of their function. 
These studies, combined with our studies of the 
nicotinic acetylcholine receptor, are likely to 
demonstrate that protein phosphorylation of neu- 
rotransmitter receptors is a primary mechanism 
in the regulation of synaptic transmission. 
204 
