Dr. Niznik's Laboratory
The
major research direction in the Molecular Neurobiology lab during the
past year was focused on the interaction of dopamine receptor/transporters
with other proteins in the central nervous system and their functional
role in the pathological/physiological conditions. The following are research
projects accomplished in 1999.
Direct Protein-Protein Coupling Enables Dopamine D5 and GABAA Receptor
Cross-Talk
g-aminobutyric-acid
A [GABAA] and dopamine D1 and D5 receptors represent two struc-turally
and functionally divergent families of neurotransmitter receptors. The
former comprises a class of pentameric ligand-gated chloride channels
consisting of diverse a, b and g subunits responsible for fast inhibitory
synaptic transmission. The latter belongs to a seven trans-membrane domain
receptor super-family exerting its biological effects, primarily the activation
of adenylate cyclase, via coupling to G-protein signaling cascades.
The
modulation of GABAA receptor mediated synaptic activity by dopamine D1/D5
or other neurotransmitter receptors is thought to only occur following
the activation of their respec-tive intracellular signal transduction
pathways. We report that dopamine D5, but not D1, receptors complex with
GABAA receptors in hippocampal neurons and co-transfected cells through
the direct binding of the D5 carboxyl terminal domain with the second
intracellular loop of GABAA receptor g2 subunit. Agonist dependent dopamine
D5 and GABAA receptor complex formation was obligatory for the functional
expression and maintenance of reci-procal receptor cross-talk, as indexed
by dopamine D5, but not D1, attenuated GABAA receptor-mediated whole-cell
or miniature inhibitory post-synaptic currents, and GABAA receptor stimulated
reductions of D5, but not D1, cAMP accumulation.
Direct
protein-protein binding between domains of structurally and functionally
divergent receptor classes highlights a previously unappreciated signal
transduction mechanism, whereby subtype selective G-protein coupled receptors
may dynamically regulate synaptic strength, and provides a mechanistic
framework to view aberrations of both these receptor systems in the expression
of neuropsychiatric disease states, such as schizophrenia. Manuscript
based on this work has been published in Nature 403: 274-280, 2000.
Direct Binding and Functional Coupling of a-synuclein to the Dopamine
Transporter Accelerates Dopamine-Induced Apoptosis
Mutations
in a-synuclein, a protein highly enriched in presynaptic terminals, has
been implicated in the expression of familial forms of Parkinson's disease
[PD] while native a-synuclein is a major component of intra-neuronal inclusion
bodies characteristic of PD and other neurodegenerative disorders. Although
over-expression of human a-synuclein induces dopaminergic nerve terminal
degeneration, the molecular mechanism by which a-synuclein contributes
to the degeneration of these pathways remains enigmatic.
The
data achieved in this lab over the last two years has shown that a-synuclein
complexes with the presynaptic human dopamine transporter [hDAT] in both
neurons and co-transfected cells through the direct binding of the non-Ab
amyloid component of a-synuclein to the carboxyl terminal tail of the
hDAT. a-synuclein-hDAT complex formation facilitates the membrane clustering
of the DAT, thereby accelerating cellular dopamine uptake and dopamine-induced
cellular apoptosis.
Since
the selective vulnerability of dopaminergic neurons in PD has been ascribed,
in part, to oxidative stress as a result of the cellular over-accumulation
of dopamine or dopamine-like molecules by the pre-synaptic DAT, these
data provide some mechanistic insight into the mode by which the activity
of these two proteins may give rise to this process. A manuscript has
been submitted to Nature Neuroscience for publication.
Carboxyl
Tail Substitution Transforms Agonist Affinity and Constitutive Activity
Profile of the D5 Receptor to Mimic D1 Receptors
The mammalian dopamine D1 receptor family contains two members, D1/D1A
and D5/D1B, that share high structural and pharmacological similarity.
The D5 receptor generally can be distinguished from the D1 receptor by
its higher affinity for agonists and agonist-independent constitutive
activity of adenylate cyclase. A PCR-based strategy was used to create
substi-tuted carboxyl terminal [CT] chimeras between D1 and D5 receptors
to investigate the potential role amino acid sequences within this region
may play in maintaining subtype differences. D1/D5CT chimeras expressed
in COS-7 cells retained a pharmacological profile identical with the wtD1
receptor. D5/D1CT chimeras, however, displayed agonist affinity identical
to either wtD1 or D1/D5CT chimeras, and significantly lower affinities
than observed for wtD5. Antagonist affinities for both chimeras remained
relatively unchanged from the parent receptor. The addition of the D1
carboxy terminus onto the D5 receptor also altered the constitutive activity
of the D5 receptor.
These
results suggest that specific regions within the D5 receptor carboxyl
terminus are required to maintain its high-affinity agonist binding and
constitutive activity. Manuscript based on this project has been accepted
by the Journal of Biological Chemistry for publication.
Ongoing
research projects being conducted in the lab also include: (1) modulation
of dopa-mine D5 and GABAA receptor protein:protein interaction by GABAA
receptor associated protein-GABAREP, (2) regulation of the DAT through
interactions with accessory proteins, (3) direct protein:protein interaction
between dopamine D1 receptor and NMDA receptor, and (4) cellular model
of "schizophrenic" D2 dopamine receptors.
Dr. Van Tol's
Laboratory
The
dopaminergic signaling system in the central nervous system is one of
the main targets for therapeutic intervention in a variety of psychiatric
and neurological disorders, including schizophrenia, bipolar disorder,
Huntington's disease and Parkinson's disease. One of
the main goals of our research is to obtain a complete understanding of
the individual components of the dopaminergic signaling system, so that
we can evaluate the contribution of this system to the development of
disease, improve therapeutic intervention and minimize treat-ment side-effects.
Most important in our pursuits to understand this system is the identifi-cation
cloning of five dopamine receptor subtypes. However, we are also investigating
the effectors of the dopamine signaling system, in particular, the G protein
activated inwardly rectifying potassium channels (GIRK). The research
conducted by our group involves a large variety of molecular, biochemical
and genetic approaches and is currently focused on the following areas.
SH3 Binding Domains in Dopamine Receptors
We
postulated that the various dopamine receptors contained so-called Src
Homology 3 (SH3) binding domains as a structural motif in the primary
amino acid sequence. Such domains are the targets for SH3 domains, which
are modular protein domains found in a variety of proteins involved in
cellular signaling. Research conducted over the last two and a half years
has now finally resulted in proof that at least the dopamine D4 receptor,
and possibly also the dopamine D3 receptor, contains such binding domains.
Considerable
progress has been made over the last six months on unraveling the role
of this domain in the functioning of this receptor. Research focused on
the functional role of these SH3 binding domains in the dopamine D4 receptor
revealed that the binding domain in this receptor is involved in receptor
internalization.
GIRK Channel Assembly
GIRK
channels are the effectors of various G protein coupled receptors, including
dopamine receptors. The physiological importance of these channels lies
in their ability to maintain the resting membrane potential of the cell
and thus to regulate the excitability of the cell. This group of potassium
channels consists of the assembly of a tetrameric structure of individual
GIRK subunits, of which four different units have been isolated to date.
The combination of subunits within the tetrameric channel structure determines
various physio-logical features of these channels. Our research is focused
on which factors guide the assembly of the channel, following the hypothesis
that GIRK channel subunits contain recognition sequences that guide selective
channel assembly. Major accomplishments in this area of research over
the last three years involve the cloning of all four human homologues
of this channel family and their functional characterization. Functional
work on the expres-sion of various combinations of the four different
subunits has given us the evidence for the existence of assembly recognition
sequences.
Transgenic Research on Dopamine Receptors
The
dopamine hypothesis of schizophrenia, which postulates that an overactive
dopamine system forms the basis for various aspects of the disease, constitutes
an important aspect in the thinking of the etiology and treatment of this
disorder. Various lines of evidence have suggested that in schizophrenia
the number of dopamine receptors is increased. In order to mimic this
situation in animal models to study the potential contribution of dopamine
receptor overexpression in animal models of schizophrenia, we developed
recombinant adeno-viruses that enabled us to transfer these genes into
the brains of rats. In vitro experiments demonstrated that these viral
vectors could indeed transfer high levels of dopamine receptor expression
to cells in an efficient manner for a prolonged period of time. Experiments
in which the recombinant viral vector was directly injected into rat brains
also demonstrated these vectors could introduce expression of the dopamine
receptors in vivo.
Currently,
experiments are being evaluated in which we explored the time-course of
expression of the transgene and simple locomotor paradigms in relation
to the in vivo expression of the dopamine receptor transgene. Besides
the use of adenoviral vectors, we have employed transgenic strategies
involving homologous recombination strategies in embryonic stem cells
to create transgenic mice with altered expression of dopamine receptors
in the germ line (see Transgenic Centre).
Other
areas of research in which considerable progress was made this year include
structure-activity studies on a variety of dopamine D4 receptor specific
ligands, development of a program on the genetic analysis of the dopamine
system in C. elegans, and the development of a phage-display system to
isolate peptidergic ligands for receptors.
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