Functions
of Signaling Machinery
in the Postsynaptic Density
Work
on the structure and organization of signaling complexes at the
postsynaptic membrane has given us hints about the potential structures
and functions of signaling complexes regulated by activation of
the NMDA receptor. However, there are still many intriguing
"holes" in our understanding. We are studying the
functions of two prominent molecules that are part of the NMDA receptor
complex at most glutamatergic synapses in the CNS. Both of
these molecules were first identified in the PSD fraction, cloned,
and sequenced by our lab.
SYNGAP
The
first is SynGAP, a Ras GTPase activating protein that is highly
localized to the postsynaptic density, or to small vesicles in the
cell body and dendrites that appear to be transport vesicles.
SynGAP is highly brain specific. In the PSD fraction, it exists
as four closely related splice variants, all made from one gene.
Most of the splice variants associate tightly with PSD-95 via its
PDZ domains.
SynGAP
is rapidly and stoichiometrically phosphorylated by CaM kinase II
at two sites. We are studying the effect of this phosphorylation
on SynGAP's location and enzymatic activity. We have created
mutant mice that are missing SynGAP. These mice die 2 to 4
days after birth. We are comparing synaptic function in neurons
cultured from mutant or wild type animals.
Densin
The
second molecule that we are focusing our attention on is densin-180.
We hypothesize that densin may help to localize specific signaling
molecules near their targets in the postsynaptic density.
Densin is a transmembrane protein that has a "leucine-rich
repeat" interaction domain at its extracellular N-terminus,
followed by a long mucin-like region that is highly glycosylated
and contains poly-sialic acid. The transmembrane domain is
located in the carboxyl half of the protein, and is followed by
a short membrane proximal segment, then an N-terminal PDZ domain.
We found that the cytosolic portion of densin forms a ternary complex
with actinin (an actin-associated protein) and CaM kinase II, an
important signaling molecule at the postsynaptic site. We
are looking for other proteins that associate with densin, and are
creating mutants that we hope will give us insight into densin's
function.
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