that were observed to phosphorylate the receptor 
in isolated postsynaptic membranes from Torpedo. 
Agents that activate cAMP-dependent protein kinase 
(such as forskolin and cAMP analogues) or agents 
that activate protein kinase C (such as phorbol es- 
ters) have been shown to regulate the phosphory- 
lation of the nicotinic receptor in primary muscle 
cell cultures. In contrast, although the receptor is 
phosphorylated on tyrosine residues in muscle 
cells, it is phosphorylated to a very low level, and it 
is not known how this phosphorylation is regu- 
lated. 
Dr. Huganir and his colleagues have used muscle 
cell cultures to investigate the neurotransmitters, 
hormones, and neuropeptides that modulate nico- 
tinic acetylcholine receptor phosphorylation. Re- 
cently a neuropeptide, calcitonin gene-related pep- 
tide (CGRP), has been found to be a cotransmitter 
with acetylcholine at the neuromuscular junction. 
CGRP regulates the phosphorylation of the acetyl- 
choline receptor in a manner identical to that of 
agents that activate cAMP-dependent protein ki- 
nase. These results suggest that CGRP regulates 
acetylcholine receptor phosphorylation by the acti- 
vation of cAMP-dependent protein kinase and that 
CGRP may be a physiological modulator of nico- 
tinic acetylcholine receptor function at the neuro- 
muscular junction. 
Recent studies have suggested that the first mes- 
senger that regulates protein kinase C phosphory- 
lation of the acetylcholine receptor may be acetyl- 
choline itself Acetylcholine has been found to 
regulate receptor phosphorylation in an identical 
manner to that of phorbol esters, potent activators 
of protein kinase C. The molecular mechanism of 
the activation of protein kinase C by acetylcholine 
is not yet clear, but it may be that calcium ions that 
permeate the nicotinic receptor channel directly or 
indirectly activate protein kinase C. 
The first messengers that regulate the tyrosine 
phosphorylation of the nicotinic receptor are not 
known. However, recent studies in Dr. Huganir's 
laboratory have suggested that the presynaptic neu- 
ron is intimately involved in the activation of the 
protein tyrosine kinase. When immunocytochemi- 
cal techniques with antibodies to phosphotyrosine 
were used, it was found that in intact rat dia- 
phragm, the nicotinic acetylcholine receptor is 
highly phosphorylated on tyrosine residues. More- 
over, denervation of the muscle leads to a progres- 
sive decrease in phosphotyrosine phosphorylation. 
In addition, during development, tyrosine phos- 
phorylation of the receptor does not occur until 
after innervation of the muscle. These results 
strongly suggest that something from the nerve, ei- 
ther a diffusible substance or the physical contact 
of the nerve terminal itself, activates the tyrosine 
phosphorylation system. 
II. Functional Effects of Protein Phosphorylation of 
the Nicotinic Acetylcholine Receptor. 
To study the functional effects of nicotinic recep- 
tor phosphorylation. Dr. Huganir's laboratory has 
examined how phosphorylation of the receptor al- 
ters its ion channel properties. These studies were 
performed using purified preparations of the re- 
ceptor reconstituted in phospholipid vesicles. 
These results demonstrated that phosphorylation 
of the 7- and 8-subunits of the receptor by cAMP- 
dependent protein kinase increases the rate of de- 
sensitization of the receptor. Desensitization is a 
common property of all receptors and is the pro- 
cess by which a receptor is reversibly inactivated in 
the presence of its agonist. Patch-clamp techniques 
were used to analyze the single-channel properties 
of the reconstituted receptor phosphorylated on ty- 
rosine residues. The results have demonstrated that 
tyrosine phosphorylation of the p-, 7-, and 8-sub- 
units of the receptor also increases the rate of de- 
sensitization of the receptor and suggest a wide- 
spread role for tyrosine phosphorylation in the 
modulation of synaptic function. 
The functional effects of phosphorylation of the 
receptor are also being studied by site-specific mu- 
tagenesis of the phosphorylation sites on the recep- 
tor subunits. All of the known phosphorylation 
sites have been mutated, and the mutant subunits 
have been expressed in Xenopus oocytes. Expres- 
sion of the mutant subunits produces a fully assem- 
bled receptor with a normal subunit stoichiometry 
of a^P^B, although the receptor subunits are not 
phosphorylated. In collaboration with Dr. Gary 
Yellen (HHMI, The Johns Hopkins University), the 
mutant receptors have been shown to be func- 
tional by using intracellular recording techniques 
as well as single-channel recording techniques. The 
desensitization kinetics of the mutant receptors are 
currently being analyzed and compared with the ki- 
netics of desensitization of normal receptors. 
The results from these studies suggest that phos- 
phorylation of the nicotinic acetylcholine receptor 
by various protein kinases integrates the effects of 
several convergent regulatory pathways on cholin- 
ergic synaptic transmission. Moreover, protein 
phosphorylation of neurotransmitter receptors and 
ion channels may be the major molecular mecha- 
Continued 
494 
