To elucidate the mechanism of agrin's regulation 
of tyrosine phosphorylation of the AChR, Dr. Hu- 
ganir and his colleagues have been studying the pro- 
tein kinases and phosphatases that phosphorylate 
and dephosphorylate the AChR. To identify the 
protein-tyrosine kinase (s) that phosphorylates the 
receptor, they have used molecular cloning tech- 
niques. Polymerase chain reaction (PGR) tech- 
niques have been used to generate probes and iso- 
late cDNA clones from Torpedo electroplax, a tissue 
highly enriched in the AChR. Several cDNAs for 
protein-tyrosine kinases were isolated, two of which 
are highly expressed in the electric organ. Using an- 
tibodies to these two abundant protein-tyrosine ki- 
nases, Dr. Huganir's laboratory was able to show that 
these kinases represented 60% of the organ's 
protein-tyrosine kinase activity. The two kinases are 
being characterized in greater detail to see whether 
they phosphorylate the AChR and whether their ac- 
tivity is regulated by agrin. 
The level of tyrosine phosphorylation of the AChR 
may also be regulated by protein-tyrosine phospha- 
tases that dephosphorylate the receptor. Using tyro- 
sine phosphorylated AChR as a substrate, Dr. 
Huganir and his colleagues have identified the 
protein-tyrosine phosphatase in the Torpedo elec- 
tric organ that dephosphorylates the AChR. They 
have recently purified this protein phosphatase 
25,000-fold to homogeneity. This phosphatase is a 
43-kDa protein that appears to be a novel enzyme, 
based on distinct chromatographic properties and 
its sensitivity to known inhibitors of other protein- 
tyrosine phosphatases. Using protein-sequencing 
techniques. Dr. Huganir and his colleagues have ob- 
tained the amino acid sequence for several peptide 
fragments of the phosphatase and are now isolating 
cDNAs that encode the phosphatase. 
Regulation of Glutamate Receptors 
by Protein Phosphorylation 
Dr. Huganir's laboratory has also been studying 
the regulation of the major excitatory receptors in 
the brain, the glutamate receptors. These receptors 
play a major role in synaptic plasticity, neuronal de- 
velopment, and neurological disorders. Glutamate 
receptors have been classified, according to their 
preferred agonists, into three groups: AMPA (a- 
amino- 3 -hydroxy- 5 -methyl-4 -isoxazole propionic 
acid), kainate, and NMDA (A'-methyl-D-aspartate) . 
Dr. Huganir's laboratory has been studying the role 
of protein phosphorylation in the regulation of the 
function of all three subtypes. 
The regulation of glutamate receptors by protein 
phosphorylation has been examined using recombi- 
nant glutamate receptors expressed in mammalian cells 
and native glutamate receptors in neuronal cell cul- 
tures. Dr. Huganir's group has shown that PKA poten- 
tiates the kainate receptor subunit GluR6 by the direct 
phosphorylation of a single-serine residue in its major 
intracellular domain. In addition, they have found that 
the AMPA receptor subunit GluRl is basally phosphor- 
ylated on serine residues by an unidentified protein 
kinase and is specifically phosphorylated on tyrosine 
residues when the receptor is cotransfected with 
pp60'''*'''', a well-characterized protein-tyrosine kinase. 
Moreover, in recent studies the phosphorylation of na- 
tive and recombinant NMDA receptors has been exam- 
ined. The NMDARl subunit is directly phosphorylated 
and modulated by PKC. 
Regulation of the GABA^ Receptor 
by Protein Phosphorylation 
Dr. Huganir's laboratory is also investigating the 
regulation of the major inhibitory neurotransmitter 
receptors in brain, the GABA^ receptors. The phos- 
phorylation of native and recombinant GABA^ re- 
ceptors has been studied. The «!, /3j, and subunits 
of the GABA^ receptor have been expressed in HEK 
cells, and the effect of PKA and PKC phosphoryla- 
tion has been studied. 
Phosphorylation of the (Sj subunit on a single- 
serine residue by PKA decreases the peak GABA re- 
sponse and effects the desensitization kinetics of 
these recombinant receptors. In addition, PKC 
phosphorylates single-serine residues on the and 
72 subunits of the GABA^ receptor. Phosphorylation 
of these two serines also inhibits the GABA^ recep- 
tor. Moreover, during the cloning of the murine 
GABA^ receptor cDNAs, Dr. Huganir and his col- 
leagues cloned a novel form of the 72 subunit (72L) 
of the GABA^ receptor. This subunit is generated by 
the insertion of eight amino acids in the major intra- 
cellular loop of the 72 subunit by differential splic- 
ing of the mRNA. This eight-amino acid insert en- 
codes an additional PKC site. 
Thus receptors consisting of the aj, /3j and 72L 
subunits contain three phosphorylation sites for 
PKC — one serine on the /3i subunit and two serines 
on the 72L subunit. Phosphorylation of each of these 
sites is inhibitory, and with phosphorylation of all 
the sites, the peak GABA response of the receptor is 
barely detectable. These results demonstrate that 
the GABA^ receptor is functionally modulated by 
PKC and that differential splicing of the 72 subunit 
regulates the sensitivity of the receptor to PKC mod- 
ulation. 
Summary 
The results from Dr. Huganir's laboratory suggest 
that phosphorylation of neurotransmitter receptors 
NEUROSCIENCE 405 
