The
research program in the Molecular Pharmacology Section studies the biology
of the receptors through which neurotransmitters such as dopamine, serotonin,
opioids and other neuropeptides exert their actions. These receptors are
part of the family of G protein coupled receptors (GPCR) and are predicted
to be involved in many clinical disorders, such as anxiety, depression,
schizophrenia and drug addiction.
GPCR Biology
The
exciting development in this area of research has been our discovery of
the existence of GPCR dimers. We have demonstrated high molecular weight
oligomeric dopamine D1, D2, D3, opioid and serotonin 5HT1B receptors.
D2 dopamine receptor dimers and tetramers
have been documented in cultured cells and in the human brain. Using photoaffinity
labeling, we provided the first indication that different classes of dopamine
antagonists may have differential affinity for monomers and dimers. Agonist
activation of the D2 receptor resulted in upregulation of monomers and
dimers to the cell surface, whereas agonist activation of the D1 receptor
induced internalization of monomers and dimers. The ability of closely
related receptors to heterodimerize was examined using the serotonin 5HT1B
and 5HT1D receptors that clearly showed a preferential ability to heterodimerize
rather than homodimerize.
We
have also examined the ability of the m and d opioid receptors to interact
and demonstrate evidence for a novel and unique pharmacology. We tested
the hypothesis that peptides based on the transmembrane (TM) domains of
GPCRs could act as antagonists of the receptor from which they were derived.
Using the D1 receptor as a model, a synthetic peptide derived from TM6
of the receptor was shown to inhibit antagonist binding. The peptide also
inhibited agonist-independent and -dependent G protein coupling and adenylyl
cyclase activation.
Thus
we have shown, for the first time, that a peptide with the amino acid
sequence of the hydrophobic TM domain of the D1 receptor had a specific
antagonist effect on its function. We have extended these findings with
other GPCRs to show that this strategy has potential for selective receptor
antagonism.
Using
opioid and dopamine receptor systems, we have been able to investigate
the mechanism of receptor activation and the phenomenon of receptor desensitization.
Another highly signifi-cant finding reported is the different patterns
of desensitization of subtypes of receptors for the same endogenous ligand,
as exemplified by the D1 and D2 dopamine receptors.
These
studies further our understanding of the molecular mechanisms underlying
rapid receptor desensitization versus slower forms of desensitization.
Using confocal microscopy, we have photographed the movement of the receptors
on the cell surface following agonist exposure, and followed their movement
inside the cell away from the surface. These studies establishing the
normal physiological function of these receptors form the basis of our
ongoing studies of the receptors in disease states. A shorter carboxy-tail
splice variant of the m opioid receptor has been shown to be relatively
resistant to agonist-induced desensitiz-ation, unlike the longer form.
Since the variable region contains a potential phosphorylation site for
G protein coupled receptor kinase, we substituted alanine for threonine394
to completely abolish desensitization at 1 hour. We also showed an absolute
requirement for acidic residues preceding the phosphorylation site.
In
examining the role of these sites in receptor internalization, we have
established the involvement of a G protein-independent parallel pathway,
mediated by tyrosine kinase. These studies show for the first time that
a GPCR is a direct substrate for phosphorylation by tyrosine kinase.
Genes and Behaviour
We
have established a breeding colony of D1 dopamine receptor knockout mice.
We have shown that perception of reward in D1-/D1- mice has been abolished,
with a marked reduction in preference for alcohol and sucrose. We have
also shown, in these animals, a marked reduction of responding for substances
of abuse and in spatial learning. We have developed a colony of D3 receptor
knockout mice and D1-D3 double knockout mice with unique behavioural phenotypes,
such as markedly attenuated exploratory activity.
Novel GPCRs and Novel Ligands
We
have identified over 40 GPCRs systems, which had previously eluded pharmacological
detection. Thus far, we have isolated DNA encoding receptors that have
homologies to opioid, somatostatin, cannabinoid, galanin and the thyrotropin
releasing hormone. All of these genes are expressed in the central nervous
system. We believe that many of these receptors have exciting potential
to mediate specific brain functions because of their discrete localization
patterns and because they interact with as-yet unknown novel transmitters.
In
the past year, endogenous ligands for four of the receptors we cloned
have been identified. They are apelin (for APJ), prolactin releasing peptide
(for GPR10), melanin concentrating hormone (for GPR24) and urotensin II
(for GPR14). The discovery of these and other GPCR genes in the genome,
and their endogenous ligands, represents one of the most important tasks
in modern pharmacology.
We
have determined the structure of the human gene encoding apelin. We have
determined the distribution of apelin in various human and rat tissues,
including many brain areas and peripheral tissues. A synthetic apelin
peptide was injected intravenously into rats, which resulted in a lowering
of blood pressure. Intraperitoneal apelin injections induced an increase
in water-drinking behaviour.
Genes and Disease
We
are now searching to find mutant receptors in patients with neuropsychiatric
disorders and to express the receptor variants in cell lines to determine
the functional consequences of the mutation. We have completed the analysis
of 15 exons of the dopamine transporter gene in DNA obtained from drug-abuse
and alcoholism patients. A polymorphism in exon 2 marks an allele with
a frequency of 12 per cent in the control population, a polymorphism in
exon 9 was found in 32 per cent, and a polymorphism in exon 15 was found
in 26 per cent. Three rare polymorphisms were also found in exons 2, 6
and 8. The D1 receptor gene has proven to be very non-polymorphic, and
our study of the D1 dopamine receptor gene in 75 substance abusers and
Tourette's syndrome patients, using single stranded conformational polymorphism
analysis, revealed no significant sequence changes in the coding region
of the gene.
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