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Research Summary |
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Nearly every neuron in the brain secretes a bioactive
peptide [Fig. 1] along
with a conventional neurotransmitter such as acetylcholine or
norepinephrine. Most endocrine cells secrete bioactive peptides.
Many neurons and endocrine cells secrete several such peptides
with a blend of biological activities. Examples include the
ACTH-secreting cells of the anterior pituitary, which stimulate
cortisol secretion from the adrenal cortex, but also secrete
the opiate peptide beta-endorphin. The sister cells in the intermediate
pituitary take the same peptide precursor, proopiomelanocortin,
and make a skin darkening peptide called MSH, but little detectable
ACTH. And the proopiomelanocortin neurons in the brain make
other collections of peptides. [Fig.
2 ] How do they do it? And how can the blend of peptides
change during development? Why do many sympathetic neurons make
neuropeptide Y along with norepinephrine? These are some of
the questions that lure us forward into our studies.
Peptides seem to have preceded the 'classical' transmitters
as the nervous system developed - creatures like Hydra and Drosophila
utilize peptides to control key developmental decisions. Beginning
with our work on proopiomelanocortin
and the coordinate
biosynthesis of ACTH and the opioid peptide beta-endorphin
[Fig. 3], we have been fascinated with the effort that
neurons and endocrine cells devote to the biosynthesis, storage
and regulated secretion of peptides. As we have learned more
about the specific enzymes involved in the biosynthesis of peptides,
we have learned important questions to ask about how these enzymes
function in cells.
Years ago we began with questions of 'what peptides are made
in what tissue?' and then progressed to 'what enzymes are involved
in peptide biosynthesis?' Now we ask detailed questions about
how those enzymes function and how they get into the secretory
granules to do their jobs? And we ask how the secretory granules
are moved to the correct place in the cell to await secretion,
and why do they wait so long before secreting their contents
on command? These questions are asked using a number of model
systems--immortal mammalian cell lines grown in tissue culture,
fresh mammalian neurons and endocrine cells coaxed to develop
in culture, knockout mouse models, a variety of cells overproducing
the enzymes and binding molecules that function in peptide biosynthesis,
and a number of cell biological and molecular biological techniques
as they are needed to address the questions at hand.
One key enzyme that has been a major focus of effort has been
PAM
[Fig. 4]- peptidylglycine
alpha-amidating monooxygenase - a copper-, oxygen- and ascorbate-requiring
protein found in secretory granules. PAM has at least three
functional domains, a monooxygenase that starts the peptide
amidation reaction, a lyase that completes the amidation reaction,
and a routing domain that helps get PAM to the right places
in the cell. The first two domains are enzymes existing on the
inside of the secretory granule, while the third domain is on
the outside of the secretory granule. Some of our studies have
focused on the enzymatic properties of the two enzymes, going
as far as X-ray
crystallography and other biophysical
studies which are performed by our various collaborators
using our proteins. We also make site-directed mutant versions
of the proteins to decipher the enzymatic mechanisms. The routing
domain on the outside of the secretory granule has been
a recent focus of our studies, yielding several interesting
binding partners through the use of the yeast two-hybrid system.
So, why was it important to focus so much of our effort on the
process of peptide amidation? This seemingly trivial modification
to the COOH-terminus of peptides turns out to be essential for
the biological activity of many peptides. Hypothalamic peptides
like oxytocin and vasopressin along with neuropeptides like
substance P and gastrointestinal mediators like gastrin must
be amidated in order to affect their target tissues. By purifying
an enzyme capable of converting peptidylglycine precursors into
amidated products, we were then able to clone a cDNA encoding
this enzyme.
To our surprise, the modification requires the sequential
action of two enzymes, a monooxygenase and a lyase. The
monooxygenase
itself is called PHM, short for peptidylglycine alpha-hydroxylating
monooxygenase, and the lyase
is called PAL, short for peptidyl-alpha-hydroxylglycine
alpha-amidating lyase. PHM uses ascorbic acid (vitamin C) to
reduce the two copper atoms that are bound to its catalytic
core and molecular oxygen is the final component of the reaction.
PAL also requires a metal ion for activity; PAL contains zinc.
We have expressed the bifunctional PAM protein in soluble and
membrane forms and have purified milligram amounts of the two
separate catalytic domains, PHM and PAL. With Drs. Mario Amzel
and Sean Prigge, we were able to deduce the crystal
structure of PHM. One copper binds to the N-terminal domain
of the PHM catalytic core and the other to the C-terminal domain.
Along with structure function studies, our current efforts are
aimed at understanding what the cells that use PHM have to do
in order to provide
copper to the enzyme. Copper is an extremely toxic metal
and specific pumps and chaperones are generally used to deliver
copper to the proteins that need it. Mottled
mice lack ATP7A, one of the copper transporting ATPases,
corresponding to the human Menkes' disease. We are using these
mutant mice to better understand how neurons and endocrine cells
get copper into the lumen of the secretory pathway so that it
can be loaded onto PHM.
PHM is similar in sequence to dopamine
beta-monooxygenase, a key enzyme in catecholamine biosynthesis.
Despite their similarities, PHM and DBM have distinctly different
features. We are using insights gained from our studies of PHM
to understand better the unique features of DBM.
We are also exploiting a number of knockout animal models, from
fruit
flies incapable of making a domain of PAM, to parasites
that cause human diseases, to mice in which PAM cannot
be expressed, to mice with domains of the PAM-interactor Kalirin
deleted.
In addition, we study a rat model of electroshock
therapy, used in people with drug-resistant depression.
In addition to its catalytic domains, PAM has
non-catalytic regions. In particular, the transmembrane
domain and cytosolic domain of PAM need not be present for the
enzyme to function. The
role of these non-catalytic domains seems to be in getting
PAM to the right place in the cell so that it can do its job.
In particular, the cytosolic domain is essential for targeting
PAM to the secretory granules of pituitary endocrine cells
and for guiding PAM protein that has reached the cell surface
back into secretory granules following internalization.
After mapping several key determinants in the rather short cytosolic
domain of PAM, we identified proteins that interact with it.
One of these proteins, PCIP-2
is a protein kinase that is highly selective for PAM. The
Ser residue phosphorylated by PCIP-2 plays a role in secretory
granule targeting and in trafficking from late endosomes into
the TGN and secretory granules. Current studies are directed
to understanding the structure and function of PCIP-2.
Another PAM cytosolic domain interactor protein, Kalirin, is
a member of the Dbl family of GDP/GTP
exchange factors for small GTP binding proteins of the Rho
sub-family. The cytosolic domain of PAM binds to the spectrin-like
repeat region of Kalirin, and Kalirin
is especially abundant in neurons
[Fig. 5]. This region of Kalirin is followed by a Dbl
homology or DH domain, and a PH domain. Kalirin occurs naturally
in a variety of isoforms and this first DH/PH domain can be
followed by a PDZ-binding motif (Kalirin-7), an SH3 motif (Kalirin-8),
another DH/PH domain (Kalirin-9) or another DH/PH domain and
a putative serine/threonine protein kinase (Kalirin-12). The
various isoforms of kalirin are expressed at
different times during development and are localized to
different regions of the cell [Fig.
6]. Over-expression of Kalirin or its subdomains dramatically
alters
growth of axons [Fig. 7]
and formation of synapses, and current studies are aimed at
understanding the structure and function of Kalirin [Fig.
8].
Multiple RNA splicing variations give Kalirin over a dozen
forms, with sets of splicing patterns which differ among tissues
and between developmental time points. The
gene for Kalirin has about 60 exons [Fig.
9], very similar to the related family member Trio, which
is reported not to undergo such a variety of splicing variations.
Trio expression is much more widespread in the body than is
the expression of Kalirin, but Trio is indeed expressed in many
neurons during development, and is the major member of this
family in endocrine cells in the pituitary [Fig.
10].
A fascinating recent finding is that different domains of Kalirin
alter neuronal process growth in culture in patterns that are
specific for the Kalirin domain and for the target neuron
[Fig. 11]. Plasmids encoding
a variety of natural Kalirin splice variants and also individual
functional domains are introduced into cultured neuronal cells
by transfection, viral infection, or direct injection, and the
growth patterns of the cells are examined over time. Similar
studies are underway using endocrine cells.
The various isoforms of Kalirin are expressed at different times
during development and are localized to different regions of
the cell. We are using a variety of over-expression and anti-sense
techniques to study the roles of the different isoforms
of Kalirin during development [Fig.
12] and in mature neurons
[Fig. 13]. Chronic cocaine treatment of adult rats produces
changes in the levels of Kalirin-7 in the striatum and nucleus
accumbens and produces changes in dendritic architecture; we
are using this system to identify factors controlling Kalirin
expression and the mechanisms through Kalirin affects neuronal
function. |
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